Ceramics International 49 (2023) 22961–22969

Contents lists available at ScienceDirect

Ceramics International
journal homepage: www.elsevier.com/locate/ceramint

Osseointegration and anti-infection of dental implant under osteoporotic

conditions promoted by gallium oxide nano-layer coated titanium dioxide
nanotube arrays
Litao Yao a, b, c, 1, Abdullrahman M. Al-Bishari a, 1, Jiating Shen a, Zhen Wang a, Tingting Liu a,
Lieping Sheng b, Gang Wu c, Lei Lu a, *, Lihua Xu d, **, Jinsong Liu a
a
School & Hospital of Stomatology, Wenzhou Medical University, Wenzhou, 325027, Zhejiang, China
b
Department of Dentistry, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310058, Zhejiang, China
c
Department of Oral and Maxillofacial Surgery/Pathology, Amsterdam UMC and Academic Center for Dentistry Amsterdam (ACTA), Vrije Universiteit Amsterdam
(VU), Amsterdam Movement Science (AMS), Amsterdam, the Netherlands
d
Department of General Medicine, First Affiliated Hospital, Wenzhou Medical University, Wenzhou, 325000, China

A R T I C L E I N F O A B S T R A C T

Handling Editor: Dr P. Vincenzini The insufficient osteointegration between titanium (Ti) based dental implants and surrounding bone tissue under
osteoporotic conditions, as well as implant-associated infections usually associate with dental implant failure.
Keywords: Gallium (Ga) has drawn increasing attention due to its antibacterial ability and pro-osteogenic property. Herein,
Gallium oxide we established a facile strategy to deposit gallium oxide (Ga2O3) nano-layer on titanium dioxide nanotube (TNT)
Nanotube
arrays by magnetron sputtering. Both Ga2O3@TNT/50 W and Ga2O3@TNT/100 W specimens were prepared at
Titanium
different powers (50 W and 100 W), respectively, which all achieved a sustained release of Ga3+ ion for over 14
Magnetron sputtering
Antibacterial days. In vitro results showed that Ga2O3@TNT/50 W sample has good biocompatibility and osteogenic activity,
Osteointegration compared with TNT and Ga2O3@TNT/100 W samples. Moreover, both Ga2O3@TNT/50 W and Ga2O3@TNT/
100 W samples possess potent antibacterial properties, due to the released Ga3+ ion. Furthermore, in vivo results
indicated that the Ga2O3@TNT/50 W showed excellent in vivo antibacterial ability and promoted new bone
formation effectively compared with TNT implants in an osteoporotic rat model with Staphylococcus aureus
(S. aureus) infection. Therefore, such Ga2O3 deposition coating strategy provides a valuable guidance for the
potential future development of Ga-doped dental implants and TNT based drug loading surfaces for clinical
applications.

1. Introduction
Osteoporosis is a common systemic disease affects the elderly,
especially postmenopausal women. It is characterized by destruction of
the microstructure of bone tissue, decreased of bone mass and density
[1,2]. With the condition of osteoporosis, the absorption and atrophy of
alveolar bone on the upper and lower jaw was enhanced, resulting in a
reduced thickness of bone trabecular and increased separation of bone
trabecula [3].
Pure titanium (Ti) and its alloys are still the “gold standards” for
dental implant materials because of their excellent biocompatibility,
mechanical properties, and corrosion resistance [4]. However, the po­
tential infection and insufficient bone formation capability limits their
clinical performances [5,6]. The bone defects around dental implants
caused by osteoporosis could affect the bone integration of Ti implant
and lead to implant failure. Another major risk is bacterial colonization
and infection associated peri-implantitis, a common postoperative
complication which usually results in implant failure [7]. Hence, it is
essential to endow Ti with good antibacterial property as well as
anti-osteoclastic property [8,9].
For decades, highly ordered nanotubes with desired diameters on the
Ti and its alloy surface based on cost-effective electrochemical anodic

* Corresponding author.
** Corresponding author.
E-mail addresses: llu2@foxmail.com (L. Lu), lihuaxu@wmu.edu.cn (L. Xu).
1
Co-first authors: These authors contributed equally to this work.

https://doi.org/10.1016/j.ceramint.2023.04.121
Received 8 February 2023; Received in revised form 12 April 2023; Accepted 14 April 2023
Available online 14 April 2023
0272-8842/© 2023 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
L. Yao et al.
oxidation, namely TiO2 nanotubes (TNT), exhibit good biocompatibility,
and has been reported to be conducive to the adhesion, proliferation,
differentiation and mineralization of osteoblasts [10]. Besides, TNT can
also be used for loading and release various drugs and bioactive com­
pounds such as bioactive metal ions and functional peptides, to enhance
the therapeutic effect of implants [11–14]. Owing to its inherent
biocompatibility and drug-loading/local release function, TNTs has
received increasing attention as an ideal surface for commercialized
orthopedic and dental implant [15,16]. The key developmental chal­
lenges for the long term success of TNT based dental implants in clinical
dentistry are simultaneously ensuring the osseointegration while
improving the long-term immunomodulation, local antibacterial prop­
erty and minimized cytotoxicity [16,17]. Various bioactive metallic ions
incorporated biomaterials has enhanced interest in specific biomedical
applications including anti-inflammation, anti-infection, enhancing tis­
sue regeneration and angiogenesis for the last few decades [18,19].
Gallium (Ga), a semi-metallic element that widely used in semi­
conductor industry, has shown therapeutic effects towards various dis­
orders, such as diagnostic and therapeutic in cancer like cancer of the
lymph system, disorders of calcium and accelerated bone resorption
[20–22]. Ga3+ ions share certain similarities with Fe3+ ions, which
competes with Fe3+ in iron-binding proteins and enzymes, and disrupts
their functions in cells. For bacteria, which highly depends on the Fe3+
mediated metabolic and signaling, resulting in inhibition of several
iron-dependent redox pathways [23]. Specifically, Ga compounds have
displayed anti-inflammatory and immunosuppressive, as well as the
multi-target antimicrobial properties against multidrug-resistant (MDR)
pathogens which attracted attention of researchers to develop Ga based
non-antibiotic-dependent antibacterial strategies to combat drug resis­
tance [19,23–25]. Moreover, some studies have reported that Ga com­
pound has enhanced the effect on the activity of osteoblasts, which play
an essential role in bone remodeling [20]. Currently, intravenous gal­
lium nitrate (Ga(NO₃)₃) is widely used in treatments of osteoporosis,
bone metabolic diseases, multiple myeloma, etc. [26–28], and was
approved by the United States Food and Drug Administration (FDA) for
clinical use in 2003 [23]. However, treatments with high dose of Ga
(NO₃)₃ was usually associated with potential clinical toxicities and side
effects. Recently, it has been reported that TNT loading with Ga(NO₃)₃
successfully enhanced the anti-bacterial property and soft-tissue inte­
gration of Ti surfaces in vitro [29,30]. In contrast to Ga(NO₃)₃, gallium
oxide (Ga2O3) is a stable metallic compound with low solubility which
had been doped into bioactive glasses to improve the cancer inhibition
and osteointegration [25]. For example, a micro-arc oxidation (MAO)
coating containing TiO2, Ga2O3 and tantalum oxide (Ta2O5) on the ti­
tanium surface showed excellent cyto-biocompatibility, osteogenic
bioactivity, and corrosion resistance [31]. Thus, we hypothesized that
the sustained release of Ga3+ ions from the Ga2O3 modified TNT on the
surface of Ti implant could enhance its osteogenic and antibacterial
effects.
Fig. 1. Schematic illustration of the preparation of Ga2O3@TNT coatings with enhanced antibacterial and osteogenic properties, in vitro and in vivo.
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Magnetron sputtering is also a conventional metallic coating method
for Ti and its alloys, which can also directly form evenly distributed
nano-scaled particles inside or on the outer edge of the TNTs [15,32,33].
The thin-film coating prepared by magnetron sputtering has the char­
acteristics of uniform deposition, uniform thickness, and high bonding
strength. Herein, the electrochemical anodic oxidation and magnetron
sputtering were used to modify the Ti surface. The Ga2O3 coated TNT
drug loading system (Ga2O3@TNT) was constructed by combining
Ga2O3 with TNT via magnetron sputtering. Furthermore, Ga2O3 reduced
the diameter of TNT, promoting sustained release of Ga3+ ions, which
has both antibacterial and osteogenic properties (Fig. 1).
2. Materials and methods
2.1. Sample preparation
Pure Ti samples (99.99%) were laser cut into sizes of 10 mm × 10
mm × 0.5 mm, polished with sandpaper of 300, 500, 1000 grits. The
TNT samples were prepared through anodization at an operating voltage
of 20 V for 1 h at 37 ◦ C. The anode of the DC power supply was con­
nected with a pure Ti sheet fixed by an electrode clamp, and the cathode
was connected with a pure platinum sheet. Then, TNT was coated with
Ga2O3 through magnetron sputtering. The control power was 50 W and
100 W for 25 min, respectively, the argon pressure was constant at 0.5
Pa, the distance between TNT and the target was 10 cm, and the tem­
perature was constant at 40 ◦ C. After sputtering was over, the sample
was taken out, vacuum-dried, and stored.
2.2. Surface characterization
The morphology of substrates was conducted using scanning electron
microscopy (SEM, S-4800, Hitachi, Japan) and transmission electron
microscopy (TEM, JEM-2100 F, JEOL, Japan). X-ray photoelectron
spectroscopy (XPS, Model PHI 5400, PerkinElmer, USA), and energy-
dispersive X-ray spectroscope (EDS, S-4800, Hitachi, Japan), attenu­
ated total reflectance Fourier transform infrared spectrometer (ATR-
FTIR, Tensor II, Bruker, Germany) were used to identify chemical
components. The phase composition and water contact angle were
measured using an X-ray diffraction (XRD, D/Max 2500 PC, Rigaku,
Japan) and contact angle goniometer (SDC-200 S, Sindin, China),
respectively.
2.3. The release profile of Ga3+
Five pieces of the Ga2O3 modified samples (Ga2O3@TNT/50 W and
Ga2O3@TNT/100 W) were soaked in 15 mL of PBS solution, respec­
tively. The samples were fetched into a new PBS solution at 1-, 4-, 7-, and
14-day time points. Finally, the collected liquid is accessed by induc­
tively coupled plasma mass spectrometry (ICP-MS, Agilent 7500,
L. Yao et al.
Agilent).
2.4. In vitro osteogenesis assays
2.4.1. Cell culture
Pre-osteoblastic MC3T3-E1 cells were used to evaluate the biocom­
patibility of materials and promote osteoblast function. The cells were
cultured in an α-MEM medium, supplemented by 10% fetal bovine
serum (FBS) and 1% penicillin/streptomycin (P/S). MC3T3-E1 cells
were stored at 37 ◦ C in a humidified incubator under 5% CO2.
2.4.2. Cell morphology
Both SEM and fluorescence microscope (FM) was used for cell
morphology observation. After the MC3T3-E1 were cultured at density
(1 × 103 cells/well) for 3 days, different samples were fixed in 4%
paraformaldehyde. Samples were dehydrated by gradient ethanol,
sprayed with gold before SEM observation. For FM morphology obser­
vation, cells were stained by fluorescein isothiocyanate (FITC) labeled
phalloidin and 4′ ,6-diamidino-2-phenylindole (DAPI), respectively
before analysis (OLYMPUS IX71, Japan).
2.4.3. Cell proliferation
After 1, 4, and 7 days of osteogenic induction at density of 2 × 104
cells/well with the α-MEM medium, the MC3T3-E1 cell viability on each
sample was tested using the MTT assay. After gently washing with PBS,
300 μL of incomplete medium and 30 μL of MTT solution was added to
each sample and incubated at 37 ◦ C for 4 h. Then, 1 mL of DMSO was
used to dissolve the formazan, and the optical density was measured
using a microplate reader (Bio-Rad 680, USA) at a wavelength of 490
nm.
2.4.4. ALP activities
Samples were sterilized with 75% ethanol and ultraviolet disinfec­
tion. MC3T3-E1 cells were seeded on different substrates at density of 2
× 104 cells/well. After 7 days of osteogenic induction, the cells cultured
on the sample were washed 3 times with PBS and fixed with 4% para­
formaldehyde for 40 min. Then samples were stained with BCIP/NBT
ALP color kit (Beyotime, China) for 40 min. Five random views were
selected to photograph by using the stereomicroscope.
For quantitative experiments, after 7 and 14 days of culturing, the
cells on the surface of different samples were lysed by 1% Triton X-100
on ice for 40 min of osteogenic induction, respectively. ALP activity was
evaluated using the ALP assay kit (Nаnjing Jiаncheng Bioengineering
Institute, China) and the BCA protein assay kit (Beyotime, China).
Finally, the total intracellular protein content determined by the BCA
protein assay kit was used to standardize the ALP results.
2.4.5. Mineralization
MC3T3-E1 cells were seeded on different substrates with the α-MEM
medium at density of 2 × 104 cells/well. After 14 days for osteogenic
induction, alizarin red S was used to stain the fixed samples for 60 min.
Five random views were selected using the stereomicroscope to observe
the mineralized nodules.
In the quantitative experiment, the mineralized nodules on the
sample were dissolved by 1 mL of cetylpyridinium chloride solution
(10%). And the absorbance of the resulting solution was measured by
using a microplate reader (Bio-Rad 680, USA) at a wavelength of 540
nm.
2.4.6. RT-PCR
MC3T3-E1 cells were seeded on different substrates with the α-MEM
medium at density of 3 × 104 cells/well. After 14 days of cell culture, the
RNAiso extraction kit is used to obtain total mRNA, and the RNA con­
centration was determined by Nano-drop 2000. Then, PrimeScriptTM
RT kit was used to reverse transcribe mRNA into cDNA. Bone formation-
related genes ALP, OCN, OSX, OPG, RunX2, COL I, and GAPDH as the
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internal reference gene for standardization were detected by SYBR
Premix ExTM TaqII kits (Takara, Kyoto, Japan). The primers are shown
in Table S1.
2.5. Antibacterial assays
Gram-positive Staphylococcus aureus (S. aureus) and gram-negative
Escherichia coli (E. coli) were cultured onto substrates at an initial den­
sity of 1 × 106 CFU/mL at 37 ◦ C before evaluation.
2.5.1. Antibacterial rates
After culturing for 4 h and 24 h, the adherent bacteria were collected
via a vibrate ultrasonication for 5 min. The diluted bacteria were then
seeded onto sterile agar plates for 24 h, then observed and counted. The
antibacterial rates were calculated as following:
C = (A − B)/A × 100%
(C: antibacterial rate; A: average colony number of TNT group; B:
average colony number of Ga2O3@TNT/50 W or Ga2O3@TNT/100 W
groups).
2.5.2. Live/dead staining of bacteria
After culturing for 4 h and 24 h, S. aureus and E. coli on different
samples were fixed with 4% paraformaldehyde and stained using a
LIVE/DEAD Baclight™ Bacterial Viability Kit (thermos fisher, USA). The
stained bacteria were then observed using FM. Five visual fields were
randomly selected for fluorescence observation.
2.5.3. Bacterial morphology
After 24 h of incubation, S. aureus and E. coli on different samples
were fixed with 4% paraformaldehyde, and dehydrated by gradient
ethanol solutions, sprayed with gold, and observed using SEM.
2.6. In vivo studies
2.6.1. Preparation of osteoporotic rat bone defect model
Ovariectomy were performed to model osteoporosis as described
previously [1,2]. Briefly, female Sprague-Dawley (SD) rats (~300 g)
were anesthetized by intraperitoneal injection of 1% chloral hydrate
prior to bilateral ovariectomy operations (OVX). After 3 months, the
osteoporotic model was established. The bone density of rats was
characterized by Micro-CT, tissue section analysis, etc. All animal pro­
tocols were reviewed and approved by the Animal Ethics Committee of
Wenzhou Medical University followed the China animal management
and committee guidelines.
2.6.2. Implantation surgery and infection model
After intraperitoneal injection anesthesia with 10% chloral hydrate
(3.4 mL/kg), OVX SD rats were randomly divided into two groups. TNT
and Ga2O3@TNT/50 W samples (diameter = 1 mm, length = 10 mm)
were inserted into the cylindrical holes through articular surface of the
femur. S. aureus suspension (10 μL, 1*105 CFU/mL) carefully injected
into the cavity to prepare an osteoporosis model with a bacterial
infection, and then muscle tissue and skin were sewed. For in vivo
antibacterial analysis, Giemsa and HE staining of bone tissue was
applied after 3 days of implantation.
2.6.3. Micro-CT analysis
Samples were collected and fixed after 1 month of implantation. The
osteogenesis around implants in the OVX SD rats were observed via
Micro-CT (Skyscan 1176, Bruker, Germany) scanning. New bone volume
(mm2), bone connectivity density (Conn-Dens), trabecular width
(Tb⋅Th), and trabecular separation (Tb.Sp) were calculated.
2.6.4. Osteogenesis assessment in vivo
Samples were decalcified for 21 days using 12%
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ethylenediaminetetraacetic acid (EDTA, pH 7.4). After implants were
carefully removed, samples were dehydrated in graded ethanol solutions
and embedded in paraffin, followed by hematoxylin and eosin (HE) and
Masson’s trichrome (MT) staining.
2.7. Statistical analysis
All experiments had at least 3 repeats, and quantitative data were
given as mean ± SD. The data was evaluated and quantified using one-
way analysis of variance (ANOVA) and Tukey’s multiple comparison test
(GraphPad Prism 7.0, San Diego, CA, USA) with a 95% confidence level.
3. Results and discussion
Magnetron sputtering technique has been widely used for thin-film
deposition associated applications, such as manufacture of semi­
conductors and the surface modification of dental implants [34,35]. It
has recently reported that TNT loading with Ga(NO₃)₃ could signifi­
cantly enhanced the antibacterial property of Ti implants in vitro [29,
30]. Compared with Ga(NO₃)₃, Ga2O3 has a much lower solubility and
higher stability which may benefits the sustained release of Ga3+ ions
while reduces its initial burst release [25]. In this study, Ga2O3 was
uniformly deposited on the surface of TNT by magnetron sputtering, and
the coating thickness was controlled by the constant deposition time for
25 min, and power range from 50 to 100 W [36].

Fig. 2. Characterization of TNT, Ga2O3@TNT/50 W and Ga2O3@TNT/100 W. (a) SEM and TEM images; (b) EDS mapping spectra; (c, d) XPS spectra; (e) water
contact angle; and (f) in vitro Ga3+ release of samples. The significance was evaluated for * p < 0.05, **p < 0.01, ***p < 0.001, and n.s. P > 0.05.

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3.1. Surface characterization
The morphology changes of samples with different power level were
observed by SEM and TEM. Fig. 2a shows the successful preparation of
TNT arrays, and the outmost surface of TNT was uniformly coated with
scattered Ga2O3 nano-particles after sputtering. There was a slightly
change on the morphology and tube diameter at the outer edge of
Ga2O3@TNT/50 W by the deposited Ga2O3, while the tube diameter at
the outer edge was dramatically reduced in Ga2O3@TNT/100 W group.
TEM analysis reveals that the scattered Ga2O3 nano-particles were
formed inside of the nanotubes after magnetron sputtering. The nano­
layer coating does not completely seal the tube opening and preserves
the hollow structure of the TNT. As a result, such Ga2O3 nanolayer
coated TNT arrays still possess the capability of drug loading.
The surface chemical composition of the different samples was
further investigated by EDS and XPS. The EDS elemental mapping show
that the Ga element was evenly distributed on the surfaces after depo­
sition, and the content of Ga in Ga2O3@TNT/50 W and Ga2O3@TNT/
100 W groups was 0.45 ± 0.06 at% and 1.15 ± 0.12 at%, respectively
(Fig. 2b, Table S2). As shown in Fig. 2c, the representative XPS spectra
show major characteristic peaks of TiO2: Ti2p3, at 464.3 eV and 458.7
eV, and O1s at 531.0 eV and 530.0 eV, respectively [23]. The Ga peaks at
1145.2 eV and 1118.3 eV were prominent in Ga2O3@TNT/50 W and
Ga2O3@TNT/100 W, as well as the emerging O1s (Ga–O) at 530.7 eV
which indicates the successful deposition of Ga2O3 [37]. The hydro­
philicity of the TNT arrays was significantly reduced after Ga2O3
deposition (Fig. 2e), which may due to the decreased tube diameter
[38]. The XRD crystal forms of different samples were detected. The TNT
sample shows the characteristic peak of rutile and anatase, and there
was no new diffraction peak in the spectra after deposition, which in­
dicates that the thin layer of Ga2O3 has little effect on the crystal
structure of TNT (Fig. S1) [39].
3.2. In vitro Ga3+ release
As show in Fig. 2f, Ga3+ ions were mainly released in the first 7 days.
The amount of released Ga3+ ions of Ga2O3@TNT/100 W was 1.5 times
higher than Ga2O3@TNT/50 W in the first 7 days, which may due to the
higher amount of Ga2O3 on sample surface. After 7 days, both samples
achieved a plateau in Ga3+ ion release. This might be because of the
diffusion barrier that forms or because of the depletion of Ga2O3 on the
sample surface. The release profile of Ga3+ ions from surfaces per time
and surface area was also shown in Fig. S2. These results show that the
release of Ga3+ ions from the samples can be managed by changing the
amount of Ga2O3 on the surface of the samples.

Fig. 3. In vitro evaluation of osteogenesis. (a) FM images of MC3T3-E1 cells on samples; the statistical analysis of (b) cell viability and (c) cell spreading; The activity
and distribution of ALP and mineralization level of each sample: (d) images (white arrows: mineralized nodules); statistical analysis of ALP activity (e) and
mineralization; (f) osteogenic differentiation related RT-PCR results. The significance was evaluated for * p < 0.05, **p < 0.01, ***p < 0.001, and n.s. P > 0.05.

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3.3. In vitro osteoblast assays and OCN. These results indicate that the Ga2O3@TNT/50 W samples
effectively enhanced the osteogenic differentiation, which may due to
3.3.1. Osteoblast morphology and viability the reduced tube diameter and released Ga3+ ions. Moreover, it seems
The FM and SEM results showed that Ga2O3@TNT/50 W signifi­ that COL-I was the main marker enhanced by the released Ga3+ ions,
cantly promoted MC3T3-E1 cell spreading compared to the TNT and the which was corresponding to previous studies [46,47].
Ga2O3@TNT/100 W groups (Fig. 3a), this may due to the additional cell
binding site from nano-scaled Ga2O3 particles on surface [40–42].
3.4. Antibacterial assessment
The cell viability of Ga2O3@TNT/50 W group after 3 and 7 days
increased significantly compared to Ga2O3@TNT/100 W group. How­
Ga3+ and its compounds are novel broad-spectrum metallic antimi­
ever, there was no significant difference in cell viability between TNT
crobials. Their pharmacological properties rely on Fe atom mimicry,
and Ga2O3@TNT/50 W groups (Fig. 3b). Moreover, the average area
thus interference the essential enzymatic processes of bacterial infection
and aspect ratio of adhered cells on Ga2O3@TNT/50 W samples are
that dependent on Fe [42,48–50]. The antibacterial performance of
significantly higher than they are on the Ga2O3@TNT/100 W samples
TNT, Ga2O3@TNT/50 W and Ga2O3@TNT/100 W samples was tested
(Fig. 3c). The inhibited cell proliferation and cell spreading of
against Gram-positive strain S. aureus and Gram-negative strain E. coli,
Ga2O3@TNT/100 W may attributed to the destruction of nanotube
which are commonly associated with implants infection.
structure [43], and the excessive released Ga3+ ions, which could affect
The morphology changes of S. aureus and E. coli after 24 h of incu­
cell viability [35,44].
bation was determined by SEM (Fig. 4a). Compared with the TNT group,
the number of adherent bacteria on Ga2O3@TNT/50 W and
3.3.2. Osteogenic differentiation
Ga2O3@TNT/100 W was reduced, which may be attributed to the
The early osteogenic differentiation of MC3T3-E1 cells was investi­
release of Ga3+ ions. In TNT group, the surfaces of spherical S. aureus
gated by measuring their ALP activity, the early indicator for osteogenic
and rodlike E. coli are smooth and damage-free. However, for
differentiation. The activity and distribution of ALP (7 days, Fig. 3 d and
Ga2O3@TNT/50 W and Ga2O3@TNT/100 W groups, the morphologies
e) and mineralization level (14 days, Fig. 3 d and f) in Ga2O3@TNT/50
of cells have changed significantly the appearance of rumples, dis­
W group were significantly improved than other groups after 7 days of
configuration and shrunken were observed.
incubation. However, after 14 days of osteogenic induction, the ALP
Fig. 4b–d shows the colonies of S. aureus and E. coli E. coli after 4 and
activity of Ga2O3@TNT/50 W was lower than TNT, which may be
24 h of culturing. TNT demonstrated non-antibacterial activity that
related to the transformation of cells to mineralization (Fig. 3e) [9,44].
bacterial colonies are all over the agar plates. However, the agar plates
Correspondingly, the mineralization level of Ga2O3@TNT/50 W group
from the Ga2O3@TNT/50 W and Ga2O3@TNT/100 W samples host
was significantly improved than other groups after 14 days of incubation
minimal bacterial colonies indicated their excellent antibacterial
(Fig. 3f).
activities.
The RT-PCR results of each sample was shown in Fig. 3g. The
Live and dead staining result showed that nearly 100% S. aureus in
Ga2O3@TNT/50 W group significantly enhanced osteogenic genes
TNT group were stained in green fluorescence, indicating they were
expression including RUNX2, OPN, and COL-I. It has been reported that
alive. However, 89.3 ± 2.5% and 94.0 ± 2.6% S. aureus were killed in
Ga promotes the expression of COL-I, which is associated with bone
Ga2O3@TNT/50 W and Ga2O3@TNT/100 W groups, respectively.
collagen formation and mineralization deposition [45]. However, the
Similar results were observed with E. coli., 92.7 ± 4.0% and 95.7 ± 2.1%
deposition of Ga2O3 had no significant effect on the expression of ALP
of bacteria was dead in Ga2O3@TNT/50 W and Ga2O3@TNT/100 W

Fig. 4. Antibacterial efficacy of TNT, Ga2O3@TNT/50 W and Ga2O3@TNT/100 W. (a) The adhesion and morphology of S. aureus and E. coli after 24 h of incubation;
(b) typical images of cultivated S. aureus and E. coli colonies from the surface of samples after 4 and 24 h of incubation; (c, d) antibacterial rates of various specimens
against S. aureus and E. coli respectively; (e) live and dead staining of adherent bacteria, and (f) the relevant statistical results. The significance was evaluated for * p
< 0.05, **p < 0.01, ***p < 0.001, and n.s. P > 0.05.

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groups, while it was almost all alive in TNT group (Fig. 4 e and f). Thus,
our research suggests that the released Ga3+ ions from Ga2O3 nano­
particles contribute to the TNT array with good antibacterial ability.
In summary, the Ga2O3@TNT/50 W group effectively promotes
osteogenic differentiation and has excellent antibacterial properties in
vitro. The Ga2O3@TNT/100 W group was discarded in subsequent in vivo
experiments due to its obvious cytotoxicity.
3.5. In vivo results
In this study, the ovariectomized (OVX) rat model was selected
because it is of great significance to the pathophysiological study of
implant osseointegration and drug treatment, which was considered as
the most suitable model for simulating postmenopausal osteoporosis by
the US Food and Drug Administration [24,51,52].
During osteoporotic conditions, the bone microenvironment effects
on the transformation of monocytes into osteoclasts, resulting in bone
loss and reduced bone density, which affects the initial stability of the
implant. The decrease in the initial stability of the implant will result in
difficulty in the formation of new bone around the implant and the
decline of the osseointegration capacity of the implant, which will
seriously affect the success rate of implant surgery.
Peri-implantitis induced by infections hurts the reconstruction of
tissues surrounding implants, which often leads to implant failure and
other serious dental diseases. The formation of plaque biofilm on the
surface of implant enhanced the resistance of bacteria to host’s defense
and antibiotics [53]. Therefore, the OVX rat model with bacterial
infection after implantation surgery was used to evaluate the effect of
released Ga3+ on antibacterial and osseointegration in vivo. In order to
check the early anti-inflammatory and osseointegration status after
Fig. 5. The therapeutical efficacy of TNT and Ga2O3@TNT/50 W implants in vivo. (a) histological images of TNT and Ga2O3@TNT/50 W after surgical implantation
for 3 days; (b) micro-CT 3D images after surgical implantation for 1 month; (c) the statistical analysis of micro-CT evaluation; (d) histological images of bone tissue
after surgical implantation for 1 month. The significance was evaluated for * p < 0.05, **p < 0.01, ***p < 0.001, and n.s. P > 0.05.
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implantation, we sacrificed the rats at 3 days and 1 month after im­
plantation, and collected femoral samples for histology analysis and
micro-CT.
3.5.1. In vivo antibacterial activity
The in vivo antibacterial property of samples was evaluated by
osteoporotic rat model with S. aureus infection. As shown by Giemsa
staining in Fig. 5a, many bacteria were distributed around the TNT
implant (red arrow). However, there was no apparent bacterial aggre­
gation observed around the Ga2O3@TNT/50 W implant, indicating that
its early antibacterial property. We speculate that the rapid release of
Ga3+ ions in the first 3 days around the implant leads to abnormal
bacterial metabolism and affects bacteria proliferation and survival due
to the competitive inhibition of the uptake of Fe3+ ions by bacteria. On
the other hand, the high surface-to-volume ratio of Ga2O3 nanoparticles
could physically destroys the bacterial membrane as well [54].
It was observed by HE staining results that many monocytes and
other inflammatory cells were aggregated around the TNT implant
(green arrow), indicating an early severe inflammatory response
induced by infection. However, no monocyte aggregation was observed
in the bone tissue nearby Ga2O3@TNT/50 W implant, suggesting no
apparent inflammation occurred, which may due to the inhibitory effect
of released Ga3+ on macrophages and T lymphocytes [52].
3.5.2. Micro-CT results
The newly formed bone around the implants were analyzed via
micro-CT 3D images. As shown in Fig. 5b, there is an increased new bone
formation around the Ga2O3@TNT/50 W group compared to TNT
group. This observation was confirmed using bone volume measure
softwares, CTan, and CTVox. The quantified data of bone volume/total
L. Yao et al.
volume (BV/TV%), trabecular thickness (Tb⋅Th), bone connectivity
density (Conn-Dens), and trabecular space (Tb.Sp) was shown in Fig. 5c,
indicating that Ga2O3@TNT/50 W had the significant better osteoin­
ductive ability than TNT.
3.5.3. Analysis of early osseointegration tissue
Both Masson and HE staining were used to further confirm the new
bone formation around the implants. As illustrated in Fig. 5d, in Masson
staining, the blue part of the bone interface around the implant repre­
sents the immature trabecular bone, while the red part represents
mature trabecular bone. It was found that more blue part tissue, namely
immature trabecular bone tissue was distributed around the
Ga2O3@TNT/50 W implant compared to TNT implant. Moreover, the
bone interface of Ga2O3@TNT/50 W implant was significantly thicker
than TNT group. For HE staining, it showed an increased bone-like tissue
around Ga2O3@TNT/50 W implant compared to TNT implant, which
consisted with micro-CT results as well.
Micro-CT and histological analysis showed that Ga2O3@TNT/50 W
can promote new bone formation and improve implantation in the
infected osteoporosis rat model, which may due to the enhanced initial
stability of the implantation and the appropriate tissue regeneration
microenvironment. As described previous, the sustained release of Ga3+
ions played a role in anti-infection and regulation of inflammatory re­
sponses in the early stage, which is beneficial to enhance the subse­
quence osseointegration process. On the other hand, the promotion of
COL-I and other osteogenesis-related gene expression from osteoblasts
by the release Ga3+ ions from Ga2O3@TNT/50 W directly stimulates
new bone formation. In addition, it has been reported the regulation of
bone mineral and matrix properties by Ga3+ ions inhibits the activity of
osteoclasts and reduces bone resorption [24,51].
4. Conclusions
In summary, we successfully sputtered Ga2O3 nanoparticles on TNT
surface via magnetron sputtering. We found that both Ga2O3@TNT/50
W and Ga2O3@TNT/100 W had superior antibacterial ability in vitro.
Meanwhile, it was indicated that the sustained release of Ga3+ ions from
Ga2O3@TNT/50 W could significantly improve the osteogenic differ­
entiation ability of MC3T3-E1, whereas Ga2O3@TNT/100 W group
showed cytotoxicity. Moreover, in vivo experiments have confirmed that
Ga2O3@TNT/50 W group has excellent antibacterial effect and effec­
tively promotes new bone formation in an infected rat model of osteo­
porosis in comparison with TNT group. Our work provides a novel
strategy to effectively deposits Ga2O3 on the surface of Ti implants to
enhance its antibacterial and bone integration properties in osteoporotic
situations. The future studies may focus on the regulation mechanism of
such systems on osteoclasts and related immune cells, exploring their
potential drug loading abilities, and improving their mechanical prop­
erties for clinical application.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work is supported by the National Natural Science Foundation
of China (81870810 and 82071170), Zhejiang Provincial Science and
Technology Project for Public Welfare (LGF21H140004,
LTGY23H140001), Wenzhou Public Welfare Science and Technology
Project (ZY2019009, 2021Y0441 and Y20210114) and Traditional
Chinese Medicine Science and Technology Program of Zhejiang Prov­
ince (2023ZR109).
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Ceramics International 49 (2023) 22961–22969
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ceramint.2023.04.121.
References
[1] Y. Jin, L. Xu, X. Hu, S. Liao, J.L. Pathak, J. Liu, Lithium chloride enhances bone
regeneration and implant osseointegration in osteoporotic conditions, J. Bone
Miner. Metabol. 35 (2017) 497–503.
[2] J. Liu, J.L. Pathak, X. Hu, Y. Jin, Z. Wu, M.A. Al-Baadani, S. Wu, H. Zhang,
S. Farkasdi, Y. Liu, Sustained release of zoledronic acid from mesoporous TiO2-
layered implant enhances implant osseointegration in osteoporotic condition,
J. Biomed. Nanotechnol. 14 (2018) 1965–1978.
[3] J.S. Khurana, L.A. Fitzpatrick, Osteoporosis and Metabolic Bone Disease, Bone
Pathology, Springer, 2009, pp. 217–237.
[4] R. Rasouli, A. Barhoum, H. Uludag, A review of nanostructured surfaces and
materials for dental implants: surface coating, patterning and functionalization for
improved performance, Biomater. Sci. 6 (2018) 1312–1338.
[5] A. Czan, O. Babík, M. Miklos, L. Záušková, V. Mezencevová, Assessment of surface
area characteristics of dental implants with gradual bioactive surface treatment,
Technol. Eng. 14 (2017) 35–39.
[6] M. Rottmar, E. Müller, S. Guimond-Lischer, M. Stephan, S. Berner, K. Maniura-
Weber, Assessing the osteogenic potential of zirconia and titanium surfaces with an
advanced in vitro model, Dent. Mater. 35 (2019) 74–86.
[7] P. Augat, U. Simon, A. Liedert, L. Claes, Mechanics and mechano-biology of
fracture healing in normal and osteoporotic bone, Osteoporos. Int. 16 (2005)
S36–S43.
[8] K. Page, R.G. Palgrave, I.P. Parkin, M. Wilson, S.L. Savin, A.V. Chadwick, Titania
and silver–titania composite films on glass—potent antimicrobial coatings,
J. Mater. Chem. 17 (2007) 95–104.
[9] R.O. Darouiche, Treatment of infections associated with surgical implants, N. Engl.
J. Med. 350 (2004) 1422–1429.
[10] P. Roy, S. Berger, P. Schmuki, TiO2 nanotubes: synthesis and applications, Angew.
Chem. Int. Ed. 50 (2011) 2904–2939.
[11] G. Balasundaram, C. Yao, T.J. Webster, TiO2 nanotubes functionalized with
regions of bone morphogenetic protein-2 increases osteoblast adhesion, J. Biomed.
Mater. Res. 84 (2008) 447–453.
[12] S.-K. Min, H.K. Kang, D.H. Jang, S.Y. Jung, O.B. Kim, B.-M. Min, I.-S. Yeo, Titanium
surface coating with a laminin-derived functional peptide promotes bone cell
adhesion, BioMed Res. Int. (2013) 2013.
[13] B. Wang, A. Bian, F. Jia, J. Lan, H. Yang, K. Yan, L. Xie, H. Qiao, X. Chang, H. Lin,
Dual-functional” strontium titanate nanotubes designed based on fusion peptides
simultaneously enhancing anti-infection and osseointegration, Biomaterials
Advances 133 (2022), 112650.
[14] B. Wang, Z. Wu, S. Wang, S. Wang, Q. Niu, Y. Wu, F. Jia, A. Bian, L. Xie, H. Qiao,
Mg/Cu-doped TiO2 nanotube array: a novel dual-function system with self-
antibacterial activity and excellent cell compatibility, Mater. Sci. Eng. C 128
(2021), 112322.
[15] Y. Li, Y. Yang, R. Li, X. Tang, D. Guo, Y. Qin, Enhanced antibacterial properties of
orthopedic implants by titanium nanotube surface modification: a review of
current techniques, Int. J. Nanomed. (2019) 7217–7237.
[16] K. Gulati, S. Ivanovski, Dental implants modified with drug releasing titania
nanotubes: therapeutic potential and developmental challenges, Expet Opin. Drug
Deliv. 14 (2017) 1009–1024.
[17] D. Chopra, K. Gulati, S. Ivanovski, Understanding and optimizing the antibacterial
functions of anodized nano-engineered titanium implants, Acta Biomater. 127
(2021) 80–101.
[18] G. Janarthanan, I. Noh, Recent trends in metal ion based hydrogel biomaterials for
tissue engineering and other biomedical applications, J. Mater. Sci. Technol. 63
(2021) 35–53.
[19] V.M. Schatkoski, T.L. do Amaral Montanheiro, B.R.C. de Menezes, R.M. Pereira, K.
F. Rodrigues, R.G. Ribas, D.M. da Silva, G.P. Thim, Current advances concerning
the most cited metal ions doped bioceramics and silicate-based bioactive glasses for
bone tissue engineering, Ceram. Int. 47 (2021) 2999–3012.
[20] M.B. Aldrich, J.C. Rasmussen, C.E. Fife, S.F. Shaitelman, E.M. Sevick-Muraca, The
development and treatment of lymphatic dysfunction in cancer patients and
survivors, Cancers 12 (2020).
[21] C. Xie, P. Li, Y. Liu, F. Luo, X. Xiao, Preparation of TiO2 nanotubes/mesoporous
calcium silicate composites with controllable drug release, Mater. Sci. Eng. C 67
(2016) 433–439.
[22] Y. Dong, H. Ye, Y. Liu, L. Xu, Z. Wu, X. Hu, J. Ma, J.L. Pathak, J. Liu, G. Wu, pH
dependent silver nanoparticles releasing titanium implant: a novel therapeutic
approach to control peri-implant infection, Colloids Surf. B Biointerfaces 158
(2017) 127–136.
[23] F. Li, F. Liu, K. Huang, S. Yang, Advancement of gallium and gallium-based
compounds as antimicrobial agents, Front. Bioeng. Biotechnol. 10 (2022).
[24] Y. Kaneko, M. Thoendel, O. Olakanmi, B.E. Britigan, P.K. Singh, The transition
metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has
antimicrobial and antibiofilm activity, J. Clin. Invest. 117 (2007) 877–888.
[25] C.R. Chitambar, Medical applications and toxicities of gallium compounds, Int. J.
Environ. Res. Publ. Health 7 (2010) 2337–2361.
L. Yao et al.
[26] A. Rahimnejad Yazdi, L. Torkan, W. Stone, M.R. Towler, The impact of gallium
content on degradation, bioactivity, and antibacterial potency of zinc borate
bioactive glass, J. Biomed. Mater. Res. B Appl. Biomater. 106 (2018) 367–376.
[27] G. Lusvardi, G. Malavasi, L. Menabue, S. Shruti, Gallium-containing
phosphosilicate glasses: functionalization and in-vitro bioactivity, Mater. Sci. Eng.
C 33 (2013) 3190–3196.
[28] K.S. Rana, L.P.D. Souza, M.A. Isaacs, F.N. Raja, A.P. Morrell, R.A. Martin,
Development and characterization of gallium-doped bioactive glasses for potential
bone cancer applications, ACS Biomater. Sci. Eng. 3 (2017) 3425–3432.
[29] A. Jayasree, M.N. Gómez-Cerezo, E. Verron, S. Ivanovski, K. Gulati, Gallium-doped
dual micro-nano titanium dental implants towards soft-tissue integration and
bactericidal functions, Materials Today Advances 16 (2022), 100297.
[30] S. Maher, D. Linklater, H. Rastin, P. Le Yap, E.P. Ivanova, D. Losic, Tailoring
additively manufactured titanium implants for short-time pediatric implantations
with enhanced bactericidal activity, ChemMedChem 17 (2022), e202100580.
[31] F. Wang, X. Wang, E. Xie, Q. Gan, S. Ping, J. Wei, F. Li, Z. Wang, Simultaneous
incorporation of gallium oxide and tantalum microparticles into micro-arc
oxidation coating of titanium possessing antibacterial effect and stimulating
cellular response, Biomaterials Advances (2022), 212736.
[32] M. Nycz, K. Arkusz, D.G. Pijanowska, Influence of the silver nanoparticles (AgNPs)
formation conditions onto titanium dioxide (TiO2) nanotubes based electrodes on
their impedimetric response, Nanomaterials 9 (2019) 1072.
[33] C. Pan, T. Liu, Y. Yang, T. Liu, Z. Gong, Y. Wei, L. Quan, Z. Yang, S. Liu,
Incorporation of Sr2+ and Ag nanoparticles into TiO2 nanotubes to synergistically
enhance osteogenic and antibacterial activities for bone repair, Mater. Des. 196
(2020), 109086.
[34] A. Abdal-hay, N. Abbasi, M. Gwiazda, S. Hamlet, S. Ivanovski, Novel
polycaprolactone/hydroxyapatite nanocomposite fibrous scaffolds by direct melt-
electrospinning writing, Eur. Polym. J. 105 (2018) 257–264.
[35] Y. Zhu, Y. Gu, S. Qiao, L. Zhou, J. Shi, H. Lai, Bacterial and mammalian cells
adhesion to tantalum-decorated micro-/nano-structured titanium, J. Biomed.
Mater. Res. 105 (2017) 871–878.
[36] E. Verron, M. Masson, S. Khoshniat, L. Duplomb, Y. Wittrant, M. Baud’Huin,
Z. Badran, B. Bujoli, P. Janvier, J.C. Scimeca, Gallium modulates osteoclastic bone
resorption in vitro without affecting osteoblasts, Br. J. Pharmacol. 159 (2010)
1681–1692.
[37] I. Lopez, A.D. Utrilla, E. Nogales, B. Mendez, J. Piqueras, A. Peche, J. Ramírez-
Castellanos, J.M. Gonzalez-Calbet, In-doped gallium oxide micro-and
nanostructures: morphology, structure, and luminescence properties, J. Phys.
Chem. C 116 (2012) 3935–3943.
[38] Y. Li, Y. Qi, Q. Gao, Q. Niu, M. Shen, Q. Fu, K. Hu, L. Kong, Effects of a micro/nano
rough strontium-loaded surface on osseointegration, Int. J. Nanomed. 10 (2015)
4549.
[39] K. Zhang, Y. Zhu, X. Liu, Z. Cui, K.W. Yeung, H. Pan, S. Wu, Sr/ZnO doped titania
nanotube array: an effective surface system with excellent osteoinductivity and
self-antibacterial activity, Mater. Des. 130 (2017) 403–412.
22969
Ceramics International 49 (2023) 22961–22969
[40] E. Verron, J. Bouler, J. Scimeca, Gallium as a potential candidate for treatment of
osteoporosis, Drug Discov. Today 17 (2012) 1127–1132.
[41] L. Todorov, I. Kostova, M. Traykova, Lanthanum, Gallium and their impact on
oxidative stress, Curr. Med. Chem. 26 (2019) 4280–4295.
[42] E. Verron, A. Loubat, G. Carle, C. Vignes-Colombeix, I. Strazic, J. Guicheux,
N. Rochet, J. Bouler, J. Scimeca, Molecular effects of gallium on osteoclastic
differentiation of mouse and human monocytes, Biochem. Pharmacol. 83 (2012)
671–679.
[43] H.C. Blair, S.L. Teitelbaum, H.L. Tan, P.H. Schlesinger, Reversible inhibition of
osteoclastic activity by bone-bound gallium (III), J. Cell. Biochem. 48 (1992)
401–410.
[44] N. Wang, H. Li, W. Lü, J. Li, J. Wang, Z. Zhang, Y. Liu, Effects of TiO2 nanotubes
with different diameters on gene expression and osseointegration of implants in
minipigs, Biomaterials 32 (2011) 6900–6911.
[45] H. Yu, X. Huang, X. Yang, H. Liu, M. Zhang, X. Zhang, R. Hang, B. Tang, Synthesis
and biological properties of Zn-incorporated micro/nano-textured surface on Ti by
high current anodization, Mater. Sci. Eng. C 78 (2017) 175–184.
[46] H. Qiao, C. Zhang, X. Dang, H. Yang, Y. Wang, Y. Chen, L. Ma, S. Han, H. Lin,
X. Zhang, Gallium loading into a polydopamine-functionalised SrTiO3 nanotube
with combined osteoinductive and antimicrobial activities, Ceram. Int. 45 (2019)
22183–22195.
[47] F. He, C. Qiu, T. Lu, X. Shi, J. Ye, Conjunction of gallium doping and calcium
silicate mediates osteoblastic and osteoclastic performances of tricalcium
phosphate bioceramics, Biomed. Mater. 17 (2021), 015012.
[48] Y. Zhu, X. Liu, K.W. Yeung, P.K. Chu, S. Wu, Biofunctionalization of carbon
nanotubes/chitosan hybrids on Ti implants by atom layer deposited ZnO
nanostructures, Appl. Surf. Sci. 400 (2017) 14–23.
[49] L.G. Jenis, C.E. Waud, G.S. Stein, J.B. Lian, D.T. Baran, Effect of gallium nitrate in
vitro and in normal rats, J. Cell. Biochem. 52 (1993) 330–336.
[50] A. Cochis, B. Azzimonti, C. Della Valle, E. De Giglio, N. Bloise, L. Visai, S. Cometa,
L. Rimondini, R. Chiesa, The effect of silver or gallium doped titanium against the
multidrug resistant Acinetobacter baumannii, Biomaterials 80 (2016) 80–95.
[51] R. García-Contreras, B. Pérez-Eretza, E. Lira-Silva, R. Jasso-Chávez, R. Coria-
Jiménez, A. Rangel-Vega, T. Maeda, T.K. Wood, Gallium induces the production of
virulence factors in Pseudomonas aeruginosa, Pathogens and disease 70 (2014)
95–98.
[52] A. Król, P. Pomastowski, K. Rafińska, V. Railean-Plugaru, B. Buszewski, Zinc oxide
nanoparticles: synthesis, antiseptic activity and toxicity mechanism, Adv. Colloid
Interface Sci. 249 (2017) 37–52.
[53] K.R. Raghupathi, R.T. Koodali, A.C. Manna, Size-dependent bacterial growth
inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles,
Langmuir 27 (2011) 4020–4028.
[54] D. Thompson, H. Simmons, C. Pirie, H. Ke, FDA Guidelines and animal models for
osteoporosis, Bone 17 (1995) S125–S133.