Biomaterials Advances 134 (2022) 112684

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Biomaterials Advances
journal homepage: www.elsevier.com/locate/bioadv

Synergic role of zinc and gallium doping in hydroxyapatite nanoparticles to

improve osteogenesis and antibacterial activity
Mahshid Shokri a, Mahshid Kharaziha a, , Hossein Ahmadi Tafti b,
⁎
Mohamadreza Baghaban Eslaminejad c, Rouhollah Mehdinavaz Aghdam d
a
Department of Materials Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
b
Tehran Heart Hospital Research Center, Tehran University of Medical Sciences, Tehran, Iran
c
Department of Stem Cells and Developmental Biology, Cell Sciences Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
d
School of Metallurgy & Materials Engineering, Faculty of Engineering, University of Tehran, Tehran, Iran

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

Keywords: Recently, postoperative bone infections have been one of the most crucial challenges for surgeons. This study aims to
Zinc and gallium doped hydroxyapatite synergistically promote antibacterial and osteoconductive properties of hydroxyapatite (HAp) nanoparticles through
Sol-gel method binary doping of Zn2+ and Ga3+ ions (Zn-Ga:HAp). Zn-Ga:HAp nanopowders with spherical morphology and homo-
Antibacterial activity geneous size are synthesized using a simple sol-gel method. Substitution of both zinc and gallium in the structure of
Osteogenesis
HAp results in a gradual decrease in the lattice parameters as doping level increases, limits the growth of HAp particles
and reduces its crystallinity. Noticeably, the crystallinity of HAp (85%) reduces to less than 73% (for XZn = 0.1), 78%
(for XGa = 0.4) and 75% (for XZn = 0.1 and XGa = 0.4). Ion doping also significantly modulate the release of bioactive
ions (Ca2+, PO3−4 , Zn
2+
, Ga3+) from the Zn-Ga:HAp depended on the overall amount of Ga and Zn in the HAp, which
could mediate the biological responses. Incorporating both Zn2+ and Ga3+ ions in HAp structure could significantly
improve the antibacterial activity of HAp nanopowders against Staphylococcus aureus (S. aureus) and Escherichia coli
(E. coli) with a concentration-dependent effect. Noticeably, Zn-Ga:HAp (XZn = 0.1 and XGa = 0.4) powder shows
the antibacterial activity of more than 68% and 84% against E. coli and S. aureus, respectively, at the concentration
of 500 μg/ml, thereby showing excellent antibacterial properties. In addition, Zn-Ga:HAp nanopowders not only do
not exhibit any cytotoxicity towards hMSCs, but also show significantly superior osteogenic properties. For instance,
Zn-Ga:HAp (XZn = 0.1 and XGa = 0.4) nanopowders significantly enhance the alkaline phosphatase activity (approx-
imately 2-fold) and mineralization (approximately 3-fold) of hMSCs after 14 days of culture, compared to pure HAp.
Overall, Zn-Ga:HAp (XZn = 0.1 and XGa = 0.4) with desired osteogenesis and antibacterial activity compared to
pure HAp, Zn:HAp and Ga:HAp shows promising opportunities for the implant-associated infections and the efficient
healing of bone defects.

1. Introduction
Repair of bone defects and surgical complications such as infection are
significant concerns in bone surgeries [1]. If postoperative bone infections
are ignored, osteomyelitis happens which is very difficult to treat. Systemic
administration of antibiotics or topical release by antibiotic-filled bone ce-
ment also revealed limited therapeutic efficacy, since these drugs stay in the
wound for a short time [2,3]. Therefore, more efforts have been made to de-
velop new materials that can mimic bone composition and structure, and
increase bone regeneration and long-term antimicrobial properties [4–6].
Hydroxyapatite (HAp) is the major mineral part of bone tissue with a
nanometer-scale structure. Hence, comprehensive studies have focused on
the HA-based components for bone tissue engineering and surface coating
⁎ Corresponding author.
E-mail address: kharaziha@cc.iut.ac.ir (M. Kharaziha).
of implants [7–9]. However, there are different ions in the chemical compo-
sition of HAp in bone tissue including Zn, Sr, Mg, K, Ag, Ce and Cu, making
it different from synthetic ones [10,11]. These ions not only change the spa-
tial group, degradation rate and mechanical characteristics of HAp, but also
modulate the biological responses of cells. Therefore, extensive studies are
underway on the substituting various elements in the structure of HAp
[10–13]. Between different cations, zinc (Zn2+) can stimulate bone forma-
tion and osteogenic responses by increasing ossification-related gene ex-
pression, and extracellular matrix synthesis [14]. In addition, zinc ions act
a crucial role in preventing the initial adhesion of bacteria [15] . Due to
the importance of zinc and its role in the human body, the inclusion of
zinc inbioceramic structures has been extensively studied [16–19]. Zn-
doped HAp (Zn-HAp) nanoparticles have been synthesized using various
methods such as co-precipitation [20], ion-exchange [21], hydrothermal
[22] and sol-gel [23] processes. Miyaji et al. [24] synthesized the Zn-HAp
nanoparticles and found that incorporation of 15%mol Zn significantly

http://dx.doi.org/10.1016/j.msec.2022.112684
Received 30 November 2021; Received in revised form 14 January 2022; Accepted 22 January 2022
Available online 7 February 2022
0928-4931/© 2022 Elsevier B.V. All rights reserved.
M. Shokri et al.
modulates the properties of crystallinity. Bigi and colleagues [25] synthe-
sized HAp nanoparticles doped with 25%mol Zn and confirmed that in-
creasing the zinc concentration resulted in the formation of an
amorphous apatite-like phase. Chung et al. [26] demonstrated that more
than 1000 ppm of zinc was required to provide antibacterial properties
and promote cell adhesion. Ito et al. [27] showed that the replacement of
1.2 wt% zinc in the HAp structure led to the increased antibacterial potency
as well as, growth and osteogenic differentiation of mesenchymal stem cells
(MSCs). To improve the antibacterial properties of Zn2+ ions, co-doping of
various anions and cations in the structure of HAp is a new concept aiming
to use the synergistic effect of several substituent elements.
Gallium (Ga3+) is one of the most important ions in controlling immu-
nosuppressive and antibacterial activities. In bacterial growth and multipli-
cation process, gallium ions (Ga3+) are replaced by iron (Fe3+) in the
structure of enzymes, suppressing the growth of bacteria. Moreover, studies
revealed gallium provides an efficient anti-resorptive activity that is benefi-
cial against severe bone diseases such as osteoporosis and bone tumors [28,
29]. Gallium ions also promote the proliferation of osteoblasts and osteo-
genesis, hinder bone resorption, and reduce calcium concentration in
plasma. Gallium ions are easily incorporated into the HAp matrix and im-
prove the biomechanical characteristics of the skeletal system [30]. Incom-
plete studies have been conducted on the substitution of Ga3+ in the
structure of HAp. Most studies revealed excellent antibacterial properties
against a variety of bacteria and gallium antioxidant properties. For in-
stance, Kurtjak et al. [31] synthesized Ga3+-substituted HAp and studied
its effect on biological properties. They found that by controlling the
amount of Ga3+ in the HAp structure, antibacterial and osteoconductive
properties were simultaneously improved. Ballardini and colleagues [2]
also demonstrated the ability of Ga3+ to increase calcium and treat osteo-
porosis. While previous studies have examined doping HAp with Ga3+
and Zn2+, the synergistic effect of zinc and gallium ions on the HAp struc-
ture has not been widely investigated.
The aim of the present study is to synthesize HAp nanoparticles contain-
ing substituted zinc and gallium ions (Zn-Ga:HAp) by sol-gel method to ac-
celerate the bone regeneration process and control the antibacterial
behavior. In this regard, after the optimization of Zn2+ ion concentration
doped in the HAp nanoparticles (Zn:HAp), various concentrations of
Ga3+ have incorporated in it to synthesis Zn-Ga:HAp nanoparticles. Ga:
HAp nanoparticles are also synthesized similarly, to investigate the role of
Ga3+ ions. The synthesized nanoparticles are chemically and physically
characterized and the antibacterial properties against two strains of gram-
positive Staphylococcus aureus (S. aureus, ATTC 25922) and gram-negative
Escherichia coli (E. coli, ATTC 25923) are studied. Finally, the effects of sin-
gle and multiple ion doping are studied based on the cytotoxicity and oste-
ogenic potential on the human mesenchymal stem cells (hMSCs).
2. Materials and methods
2.1. Materials
To synthesize the Zn-Ga:HAp nanoparticles (NPs), calcium nitrate [Ca
(NO3)2·4H2O, 99%, Merck, Germany], phosphorus pentoxide (P2O5;
Sigma Aldrich, US, 97%), gallium nitrate (Ga(NO3)3, Sigma Aldrich, US,
99.99%) and Zn(NO3)2·4H2O (Merck, Germany, 99.99%) were used as
precursors.
2.2. Synthesis of Zn-Ga:HAp nanoparticles
In this study, sol-gel method was used to synthesis HAp-based NPs. Al-
coholic solution of 0.50 M P2O5 and an aqueous solution of 1.67 M Ca
(NO3)2.4H2O were mixed considering the molar ratio of 10:3 of Ca/P (stoi-
chiometry related to HAp). The pH value of the initial solution was approx-
imately 2, which was adjusted to 7 using ammonia solution. To synthesis of
HAp powders doped with gallium and zinc (Zn:HAp, Ga:HAp, Zn-Ga:HAp),
gallium nitrate and zinc nitrate precursors with the desired molar ratios
were added to the aqueous solution of calcium nitrate. Zn:HAp powders,
2
Biomaterials Advances 134 (2022) 112684
Ca10−xZnx(PO4)6(OH)2, were synthesized with XZn = 1, 0.5, 0.2, 0.1 and
0.05. Ga:HAp powders, Ca10−xGax(PO4)6(OH)2, were also prepared with
XGa = 0.6. Zn-Ga:HAp powders, Ca10−(XZn-XGa)ZnxGaY(PO4)6(OH)2, were
also synthesized with X = 0.1 and Y = 0.6, 0.4, 0.2. To form the gel, the
solutions were mixed at 60 °C for 1 h and placed at 80 °C for 6 h. After
room-temperature aging for 2 days, the gels were dried at 80 °C for over-
night. Finally, the dried gels were calcified at 600 °C for 3 h.
2.3. Physical and chemical characterization of Zn-Ga:HAp nanoparticles
The surface morphology and size of NPs were studied by Field Emission-
Scanning Electron Microscope (FESEM; MIRA III, TESCAN). ImageJ soft-
ware was applied to establish the average particle size of nanoparticles. In
addition, the size distribution and ζ-potential of nanoparticles were exam-
ined by Dynamic Light Scattering (DLS, Malvern Zetasizer 3000E). In
order to determine the Zeta potential of powders, they were immersed in
water for 24 h. The chemical structure of powders was investigated by X-
ray diffraction (XRD, X' Pert Pro, Phillips). The diffraction patterns were ob-
tained using KαCu radiation (λ = 0.15418 nm) in the range of 5 ° < 2θ <
100°, step size of 0.02°. The crystallite size of the synthesized doped-HAp
nanopowders was estimated from the Scherrer Eq. (1) [32]:
0:89λ
L¼ (1)
βcosθ
where L is crystallite size (nm), λ is the wavelength (nm) KαCu radiation, β
corresponds to the full width at half maximum (FWHM) for the peak hkl
(rad), θ is the diffraction angle (in degrees). Lattice parameters (a and
c) were also calculated from two prominent peaks of (200) and (002), re-
spectively. Moreover, the crystallinity degree Xc corresponding to the crys-
talline content of the HAp powders was evaluated by Eq. (2) [32,33]:
I300  V112=300
Xc ¼ (2)
I300
where I300 is the intensity of (300) peak and V112/300 the intensity of the
hollow between (112) and (3 0 0) peaks of HAp. In addition, the functional
groups of HAp powders were analyzed by Fourier-transform infrared spec-
troscopy (FTIR, Perkin Elmer Spectrum 2) and Raman spectroscopy. The
FTIR spectra of the samples in the range 400–4000 cm−1 were recorded
on KBr pellets. Moreover, Raman studies were performed under laser exci-
tation wavelength of 1064 nm, using an RFS 100 FT-Raman Bruker spectro-
photometer. In addition, the release of Ca2+, PO3− 4 , Zn
2+
and Ga3+ ions
from the HAp based nanopowders was investigated at pH 7.4 and 37 °C
using phosphate buffer solution (PBS). 10 mg of each nanopowder were dis-
persed in 20 ml of PBS and the ion release was monitored at the specific
times points (1, 3 and 7 days). A 1 ml aliquot of the PBS containing the re-
leased ions was taken out and elemental analysis of all the samples was
measured by an inductive couple plasma atomic emission spectrometer
(ICP-AES, Perkin Elmer, Optima 7300DV, USA).
2.4. In vitro antibacterial activity assay
The antibacterial activity of NPs against gram-negative E. coli (ATCC
25922) and gram-positive S. aureus (ATCC 25923) strains was evaluated.
For this purpose, the minimum inhibitory concentration (MIC) was done
using the microdilution method [34]. MIC refers to the NP concentrations
that can inhibit bacterial growth after 24 h of incubation in vitro. MIC
was examined by observing the turbidity of the wells, before and after incu-
bation and reading the light absorption of the wells by ELISA microplate
reader. Bacto tryptic soy broth (Becton Dickinson, Sparks, MD, USA) was
prepared and autoclaved as a nutrient culture medium for bacterial growth.
Then, 100 μl of culture medium was poured into each well of a 96-well
plate under sterile condition. The doped-HAp powders were weighed for
4 different dilutions of nanoparticles in culture medium (62.5, 125, 250,
500 and 1000 μg/ml) and sterilized under UV light for 20 min. The
M. Shokri et al.
bacterial suspension was prepared according to the standard McFarland 0.5
turbidity (108 CFU/ml) and 10 μl of this suspension was added to each well.
Thus, a constant number of bacteria was affected by different concentra-
tions of nanoparticles. The prepared microplates were incubated at 37 °C
for 24 h and then their optical density (OD) at 630 nm was recorded by
ELISA microplate reader ELX800 (BioTek, Winooski, VT, USA). The exper-
iment was carried out in
triplicate and the positive control was culture medium wells with bacte-
ria, negative control was culture medium wells with different concentra-
tions of doped-HAp NPs and blank was only culture medium wells.
Bacterial survival percentage was calculated according to the following
Eq. (3) [35]:
Bacterial survival rate
 
ðOD of blank þ OD of negative controlÞ
: OD of test well–  100 (3)
ðOD of positive control  OD of blankÞ
Bacterial morphology was also investigated by SEM analysis. The steril-
ized 500 μg/ml of doped-HAp powders (n = 3) was incorporated to a 24-
well microplate with 1 ml of Bacto™ tryptic soy broth. A suspension of bac-
teria with a concentration of 108 CFU/ml (equivalent to 0.5 McFarland tur-
bidity) was made and 100 μl of this suspension was added to each well. The
microplate was incubated at 37 °C for 24 h. After treatment, the supernatant
of each well was transferred to microtubes and centrifuged at 4000 rpm for
10 min. Then, the bacteria were washed and suspended in phosphate buffer
saline (PBS, Gibco, USA). Next, they were fixed with 10% formaldehyde for
3 h and washed again with PBS. The bacteria were then dehydrated in a
graded ethanol series and 10 μl of each sample was poured onto a glass
slide and dried at room temperature for 24 h. Finally, the samples were
gold-coated and observed by SEM.
2.5. In vitro cytocompatibility and osteogenic potential evaluation
To investigate the toxicity of powders and their effect on growth, prolif-
eration and differentiation of stem cell, the extraction process was per-
formed according to ISO 10993-12 standard. The nanoparticles were
weighed at the concentrations of 250, 500 and 1000 μg/ml in 24-well mi-
croplates and sterilized by UV for 20 min. They were then incubated at
37 °C and after 24 h, the medium was removed and added to the cells.
The experiment was carried out in triplicate and the culture medium with-
out the nanoparticles was also considered as a control. Human mesenchy-
mal stem cells (hMSCs, passage# 2) were isolated from human adipose
tissue after getting informed consent approved by the Biobank Research
Ethics Committee (National Institute for Health Research - Tehran
University of Medical Sciences). They were incubated in full culture
medium made of Dulbecco's Modified Eagle Medium-low glucose
(DMEM-low, Gibco) supplemented with 10 (v/v)% fetal bovine serum
(FBS, Gibco), and 1 (v/v)% penicillin/streptomycin (Gibco) in 5% CO2.
The cytotoxicity of powders was evaluated by methylthiazolyldiphenyl-
tetrazolium bromide (MTT) assay. In this regard, hMSCs with the density of
104 cells/well were seeded in the 96-well plates in full medium and incu-
bated at 37 °C in 5% CO2 for 24 h. The culture medium was removed and
100 μl of doped-HAp powder extracts were added to each well. The cells
were incubated for 24 h and 72 h in the presence of these extracts. The cul-
ture medium was then removed and 100 μl of MTT solution (0.5 mg/ml)
was added to each well. After 4 h, formed formazan crystals were dissolved
using dimethyl sulfoxide (DMSO) and the amount of light absorption in
each well was determined by ELISA reader at 545 nm. Finally, the cell via-
bility was calculated according to the following Eq. (4):
ODS
Cell viability ð%Þ ¼  100 (4)
ODC
In this equation, ODS and ODC are the optical density of the sample and
control, respectively. Osteogenic differentiation of hMSCs was also deter-
mined using alkaline phosphatase (ALP) and Alizarin-red S assays. The
3
Biomaterials Advances 134 (2022) 112684
ALP secreted from cells in the presence of doped-HAp powder extracts
was evaluated after the 3, 7 and 14 days of culture. For this experiment,
104 cells were first seeded in a 24-well plate and after 24 h, the culture me-
dium was replaced with 300 μl of the extracts containing 10% FBS. This
process was performed every three days and at the mentioned time points,
cell supernatant was collected and the amount of ALP was measured using
an automated analyzer (Hitachi, Japan) and available kit (Pars Azmoon,
Iran) according to the manufacturer's instructions. The absorption was de-
termined using ELISA reader at 405 nm.
Alizarin-red S assay was done to study calcium deposition. In this
regard, 5000 cells with 300 μl of extracts containing 10 (v/v)% FBS were
added to a 24-well plate and incubated at 37 °C and 5% CO2. At the speci-
fied time points, the medium was replaced with new extract. After 14 days,
the medium was removed from the cells and rinsed with PBS. At this time,
the cells were immobilized with 4% paraformaldehyde (Merck) for 20 min
and then stained with 40 mM Alizarin-red (Sigma, USA) solution (pH =
4.2) for 10 min. The cells were rinsed with PBS and then were imaged
using a biological light microscope (model HP31). For quantitative analy-
sis, 800 μl of 10 (v/v)% acetic acid (CH₃COOH, Merck) was poured into
each well and placed on a shaker for 30 min. After adding the contents of
each well to the microtube, they were vortexed for 30 s and placed in an
oven at 85 °C for 10 min and then on ice for 5 min. Consequently, the
microtubes were then centrifuged at 20,000 rpm for 5 min and 500 μl of su-
pernatant was poured into the new microtubes. Then, to neutralize the acid,
200 μl of ammonium hydroxide (NH₄OH, Merck, Germany) was added to
each microtube. Finally, 100 μl of the contents of each microtube was trans-
ferred to 96-well microplate wells and their light absorption was deter-
mined by ELISA reader at 450 nm.
2.6. Statistical analysis
All biological tests were performed at least three times and the results
are reported as mean ± standard deviation (SD). The data was also ana-
lyzed using one-way ANOVA analyses and Tukey's multiple comparisons
using GraphPad, Prism Software (V.5). Differences were taken to be signif-
icant for P-value <0.05.
3. Results and discussion
3.1. Characterization of Zn:HAp nanopowder
Before synthesis of Zn-Ga:HAp nanopowder, Zn:HAp (Ca10−xZnx(PO4)6
(OH)2) nanopowder consisting of various concentrations of Zn (X = 1, 0.5,
0.2, 0.1, 0.05 and 0) was synthesized to obtain the optimum Zn content.
The weight percent of doped ions is also provided in supporting informa-
tion (Table S1). XRD = patterns of Zn:HAp nanopowders are presented in
Fig. S1. The XRD pattern of pure HAp powder was in good agreement
with the standard model of HAp (JCPDS no. 09–0432). After the incorpo-
rating Zn ions in the HAp structure, the phase stability of HAp was reduced
and a new peak related to β-TCP phase was detected at 2θ = 31°. The for-
mation of the secondary β-TCP phase in the HAp structure could be helpful
to promote ion release and improve biological properties. In other words,
increasing the amount of zinc and the presence of the β-TCP phase leads
to faster degradation of the HAp. Rapid degradation of β-TCP resulted in
better vascularization and more cancellous bone formation [36]. In the
study of Yedekci et al. [37], the increase in cell proliferation was reported
with high levels of β-TCP in the structure of HA matrix. In addition, our re-
sults revealed that, the new peaks related to zinc oxide (ZnO) were also
identified in HAp powders doped with XZn = 1 and XZn = 0.5. Moreover,
it was found that the intensity of HAp peaks decreased and became wider
with increasing Zn doping. According to Table 1, the crystallinity of the
Zn:HAp nanopowders reduced from 0.84 to less than 0.68 with increasing
Zn content upon 6 wt% (XZn = 1). It was concluded that Zn doping en-
hanced the structural defects of HAp leading to reduced crystallinity. Simi-
lar results were reported in previous studies [37–39]. For instance, Yuan
et al. [40] found that the crystallinity of HAp reduced from 0.73 to 0.59
M. Shokri et al. Biomaterials Advances 134 (2022) 112684

Table 1 ions in apatite, the crystallinity of HAp decreased and the surface area of ap-
The lattice parameter, crystallinity degree and crystal size of Zn:HAp NPs and Zn- atite increased, leading to the formation of bonds with microorganisms and
Ga:HAp NPs. cell death [43,44]. Sirajunisha et al. [45] also considered the reduction of
Sample Lattice parameters L(nm) Xᴄ crystal size in the structure as a reason for increasing the antibacterial prop-
: : erties of nanopowders. According to Fig. S3, gram-positive bacteria and
a (A) c (A)
gram-negative bacteria also showed different resistance behavior against
Pure HA 9.44016 6.89670 40.10 0.847
Zn:HAp nanoparticles, which could be due to the structure of the cell
Zn:HAp(XZn = 0.0.5) 9.42735 6.89643 43.12 0.809
Zn:HAp(XZn = 0.1) 9.42730 6.89617 37.31 0.733
wall. Zn:HAp NPs had a more significant effect on S. areus and more negli-
Zn:HAp(XZn = 0.2) 9.38180 6.89276 40.30 0.750 gible impact on E. coli. This bacterium showed the lowest sensitivity and
Zn:HAp(XZn = 0.5) 9.35936 6.88141 36.88 0.704 highest resistance in all concentrations. At the highest nanoparticle concen-
Zn:HAp(XZn = 1) 9.35224 6.87160 29.16 0.678 trations, more than 85% mortality was reported for S. areus, while at the
Zn-Ga:HAp (XZn = 0.1 and XGa = 0.2) 9.38608 6.87447 35.44 0.745
same concentration, only about 60% mortality was observed for E. coli. Sev-
Zn-Ga:HAp (XZn = 0.1 and XGa = 0.4) 9.38387 6.87347 38.84 0.750
Zn-Ga:HAp (XZn = 0.1 and XGa = 0.6) 9.36822 6.86382 40.23 0.769 eral investigations have been conducted based on interaction between NPs
Ga:HAp (XGa = 0.6) 9.37837 6.87186 38.35 0.787 and living organisms [46]. The difference between the a microorganism
with negative charge and Zn2+ could result in an absorbent electromagnet
between the microbe and the NPs, attaching the NPs to the cell surface,
after incorporation of 20 mol% Zn. These changes in the chemical compo- causing cell death [46]. Ultimately, a significant number of these interac-
sition of HAp may have diverse effects on the biological properties of HAp. tions led to the oxidation of the surface molecules of the microbes and
To find the optimized Zn:HAp nanopowder for the following experi- their rapid death. Zinc also could delay bacterial cell adhesion and biofilm
ments, cytotoxicity of various Zn-doped HAp powders on the hMSCs was in- formation, which prevented a group of bacteria from being able to stabilize
vestigated using MTT assay after 24 and 72 h incubation. As shown in and multiply [47]. Also, the growth rate of bacteria in culture medium con-
Fig. S2, cell viability was tested in the presence of Zn:HAp nanopowders taining different concentrations of Zn:HAp NPs was investigated. The low-
at three different concentrations of 250, 500 and 1000 μg/ml. According est concentration of Zn:HAp NPs that inhibited bacterial growth was
to Fig. S2, cell viability higher than 80% was reported after 72 h incubation considered as the MIC. According to Fig. S3, the survival rate of both bacte-
in contact with various samples. It could be concluded that there was no ria at a concentration of 500 μg/ml of NPs was almost constant and this con-
toxic effect on the cells. However, cell viability decreased with increasing centration could be considered as MIC. Increasing the concentration of Zn:
the NP concentration. For instance, as the concentration of Zn:HAp HAp NPs in the culture medium to 1000 μg/ml did not have a significant
nanopowder increased further to 1000 μg/ml, the cell viability decreased effect on bacterial mortality.
to about 80–85%. These results are consistent with studies by Lala et al. Since Zn:HAp nanopowders with XZn = 0.1 and 0.2 showed almost the
[41]. The higher proliferation rate of cells in the presence of HAp same increase in cell proliferation, in order to substitute gallium ions and
nanopowders doped with zinc (XZn = 0.05, 0.1 and 0.2) compared to not occupy all calcium positions in the structure, XZn = 0.1 as the optimal
cells incubated with pure HAp nanopowders was obtained. The positive in- value for the synthesis of Zn-Ga: HAp nanopowders was selected in the fol-
fluence on cell survival observed for Zn:HAp nanopowders could be due to lowing steps.
the release of more Zn2+ ions, which stimulated cell proliferation at differ-
ent concentrations of nanopowders in the cell culture medium. On the other 3.2. Characterization of Zn-Ga:HAp nanopowder
hand, the increase in cell proliferation observed with increasing zinc con-
centration in the HAp structure (XZn) from 0.05 to 0.2 might be explained In the next step, Zn-Ga:HAp nanopowders consisting of various amounts
by the high concentration of β-TCP in the doped-HAp structure. Particle of doped ions were synthesized. The weight percent of doped ions is also
size was an important parameter that affects cytotoxicity [5]. By increasing provided in supporting information (Table S1). XRD patterns of Zn-Ga:
the concentration of Zn2+ ions (XZn) in the HAp structure from 0.05 to 0.2, HAp NPs consisting of various Ga3+ contents (XGa = 0, 0.2. 0.4 and 0.6)
the particle size decreased from 43 to 36 nm and the survival rate of hMSCs and constant Zn content (XZn = 0.1) is presented in Fig. 1A. To better iden-
increased. Popa and colleagues [18] also showed that by increasing the con- tify, pure HAp and Ga:HAp (XGa = 0.6) was synthesized and characterized.
centration of Zn2+ ions in HAp from 7 to 10 mol%, the particle size de- It was found that Ga-doping resulted in the formation of β-TCP phase in the
creased from 25 to 16 nm. They also found that the cytotoxicity of HAp structure. In addition, the intensity of the HAp peak reduced and they
samples on both E. coli and human hepatocarcinoma cells (HepG2) became wider by doping Ga3+ in the structure. Similar to the results of re-
depended on the particle size. Considering that the particle size decreased search by Ballardini et al. [2], Ga-doped HAp NPs provided wider peaks
with increasing zinc concentration, the highest death rate of the cells was compared to pure HAp. In other words, with the entry of foreign ions in
reported for the HAp sample, while the lowest death was reported for the the structure of the apatite, the lattice order decreased and the crystallinity
10 mol% incorporated HAp sample. decreased. The crystallinity of the pure HAp structure decreased from 84%
Inhibition of cell proliferation after treatment with Zn-doped HAp to 78% by doping Ga3+ in the structure (XGa = 0.6), which was observed in
nanopowders (XZn = 0.5 and 1) could be attributed to the formation of other studies [3,48]. The widening of the peaks could also be attributed to a
ZnO phase. It is possible that the ZnO phase caused membrane permeability decrease in crystallite size due to the effect of Ga3+ ions on the growth of
and apoptotic activity of Zn-doped HA particles, leading to cytotoxicity. In apatite crystals or the result of a change in the structure of apatite with
other words, ZnO could disrupt the cellular communication network and is the substitution of Ga3+ ions [31].
involved in DNA damage, increased expression of the apoptotic protein To investigate the lattice defects caused by co-doping Zn2+ and Ga3+
molecule, and induced programmed cell death [42]. into the HA structure, the parameters of the HA lattice unit cell, degree of
Moreover, the antibacterial activity of Zn:HAp nanopowders was also crystallinity and crystal size were determined and are presented in
investigated (Fig. S3). In our study, it was found that the degree of inhibi- Table 1. By doping Zn2+ and Ga3+ into the HAp structure, both lattice pa-
tion of bacterial growth varied depending on the type of bacteria and rameters (a and c) were reduced, compared to HAp. These results were sim-
gram-positive bacteria are more sensitive. According to Fig. S3, as the con- ilar to the results obtained by Mellier et al. [49]. They observed a linear
centration of zinc in the structure of HAp increased, the survival rate of both decrease of the a parameter and a monotonic decrease of the c parameter
bacteria decreased. It might be related to the reduced crystallinity and crys- with increasing Ga3+ value. This behavior was expected due to the differ-
tallite size and presence of ZnO phase in these nanoparticles. Metal oxides ence in ionic radius and oxidation state of gallium compared to calcium
have shown the lowest level of toxicity and therefore, the use of these com- [49]. Therefore, the Zn-HA lattice parameters changed due to the difference
ponents to fight pathogenic microbes could be a promising choice. Simi- in the ionic radius of Zn2+ (0.74 Å) and Ga3+ (0.62 Å) and Ca2+ (0.99 Å).
larly, Ofudje et al. [39] stated that with the increasing percentage of zinc According to the results, due to the smaller radius of Zn2+ and Ga3+ ions

4
M. Shokri et al.
Fig. 1. Chemical characterization of Zn-Ga:HAp NPs: A) XRD pattern of synthesized Zn-Ga:HAp nanoparticles (0 ≤ XGa ≤ 0.6 and XZn = 0.1). B) View of the local
coordination of Zn incorporated in HAp structure.
than Ca2+, the best place for substitution in the HAp structure might be Ca
positions (Fig. 1B). As long as these ions were added to the structure, both
ions were substituted at Ca positions. However, by increasing the amount of
dopants, it was possible for their excess values to be intermediate in the
structure. Therefore, during substitution, Ga3+ replaced Ca2+, and in
order to balance the structure, it was assumed that two Ga3+ ions take
the place of three Ca2+ ions, to provide a charged equilibrium.
Our results also revealed that Ga3+ doping to the Zn:HAp (XZn = 0.1)
structure resulted in a reduction in the crystallite size from 37 nm to
35 nm for Zn-Ga:HAp (XZn = 0.1 and XGa = 0.2). However, with increasing
Ga ions to XGa = 0.6, an increasing trend in the crystallite size was ob-
tained. This was probably due to the fact that by substituting Ca2+ sites
with Zn2+ and Ga3+ in the Zn-Ga:HAp (XZn = 0.1 and XGa = 0.2), the sub-
stituent space in HAp was filled. Higher doping of gallium ions (XGa = 0.4
5
Biomaterials Advances 134 (2022) 112684
and 0.6) resulted in the formation of an interstitial defect. In addition, com-
pared to the Zn:HAp (XZn = 0.1) nanopowder, the increase in the gallium
doping from XGa = 0.2 to XGa = 0.6 in the structure of Zn:HAp (XZn =
0.1) resulted in a slight increase in the intensity of peaks. As a result, the
crystallinity increased slightly from 74% for Zn-Ga:HAp (XZn = 0.1 and
XGa = 0.2) to 76% for Zn-Ga:HAp (XZn = 0.1 and XGa = 0.6).
To investigate the morphology and size of the doped-HAp nanoparti-
cles, FE-SEM images are presented in Fig. 2A. All powders consisted of
nearly uniform and agglomerated spherical particles. According to FE-
SEM images, the average particle size of various powders was estimated
and is presented in Fig. 2B. Our results revealed the average particles of
HAp decreased from 61 to 58 nm, after incorporation of Zn2+ ions. This re-
sult was similarly reported in previous studies [18,37]. In addition, the av-
erage particle size of HAp decreased to less than 55 nm, after co-doping of
M. Shokri et al.
Fig. 2. Structural characterization of Zn-Ga:HAp NPs: A) FE-SEM images, B) average particle size, C) particle size distribution and D) Zeta-potential value of HAp, Zn:HAp and
Zn-Ga:HAp nanoparticles.
Zn2+ and Ga3+ (XZn = 0.1 and XGa = 0.4) which could be explained by the
substitution of Zn2+ and Ga3 with less ionic radius in the Ca positions.
However, the average particle size slightly increased at Zn-Ga:HAp
(XGa = 0.6) NPs which could be related to the filling of Ca positions and
the placement of Ga3+ as an intermediate in the structure.
The particle size of the powders was also investigated using DLS analy-
sis. According to Fig. 2C, the average hydrodynamic diameter of HAp and
Zn:HAp powders was estimated 296 nm and 201 nm, respectively. The dop-
ing of Zn2+ and Ga3+ reduced the average particle size compared to pure
HAp powder, which was similar to the results of other studies. For example,
6
Biomaterials Advances 134 (2022) 112684
in the study of Predoi et al. [38], the mean hydrodynamic diameter of HAp,
Zn:HAp (XZn = 0.07) and Zn:HAp (XZn = 0.2) was 52 ± 0.3 nm, 48 ±
0.3 nm and 27 ± 0.4 nm, respectively, which decreased with increasing
Zn concentration.
In addition, the Zeta potential of nanopowders was also determined and
is presented in Fig. 2D. Results revealed that the Zeta potential value of HAp
nanopowder was −8.3 mV, which was significantly increased to
−33.3 mV after Zn-doping. It could be concluded that incorporation of
Zn increased the stability of HAp powder in the solution. The increase in
the Zeta potential of HAp doped with other ions is consistent with previous
M. Shokri et al. Biomaterials Advances 134 (2022) 112684

results. In the studies of Predoi et al. [38], the Zeta potential values of HAp,
Zn:HAp (XZn = 0.07) and Zn:HAp (XZn = 0.2) were − 7.83, −23.16 and
− 34.65 mV, respectively [38]. Wahba et al. [50] also showed that the
Zeta potential of pure HAp nanopowder increased from −9 to −20 mV
in cerium-doped HAp. By co-doping Zn2+ and Ga3+ ions into the HAp
structure, the Zeta potential of the pure HAp increased to −29.4 mV in
Zn-Ga:HAp (XZn = 0.1 and XGa = 0.2). Zeta potential is used to understand
and control the properties of colloidal suspensions and the stability of col-
loidal dispersion. Colloidal solutions with high Zeta potential are more elec-
trically stable due to the increased net electrical charge on the particle
surface and the greater electrostatic repulsion between the particles. On
the other hand, colloidal solutions with low Zeta potential tend to coagulate
[51]. It can be concluded that by reducing the particle size in doped NPs
and increasing the surface to volume ratio, colloidal stability increased
and thus an increase in Zeta potential was observed. As the gallium concen-
tration in Zn-Ga:HAp nanopowders increased from XGa = 0.2 to XGa = 0.6,
the Zeta potential decreased from −29.4 to −19.3 mV, respectively, which
was consistent with DLS measurements. Further increase in the Ga3+ ion
doping in the HAp structure resulted in the formation of larger NPs which
reduced the surface charge of the particles and thus affected the stability
of the colloidal system and a slight decrease in the amount of Zeta potential
was observed.
To better identify the incorporation of ions in the structure of HAp, FTIR
and Raman spectra were studied. According to Fig. 3A, FTIR spectra of all
samples consisted of the characteristic peaks related to HAp including the
peaks at 603, 566 and 475 cm−1 attributed to the flexural movements of
the phosphate bond and the peaks at 1093, 1039 and 963 cm−1 related
to the tensile movements of phosphate in the HAp crystal lattice. Moreover,
the peaks presented at 3570 and 631 cm−1 revealed the tensile movements
of the hydroxyl ions in the HAp crystal lattice [31]. Broad peaks in the ap-
proximate areas of 1550 cm−1 were also related to carbonate impurities. In
addition, the small peak appeared at 872 cm−1 was attributed to the car-
bonate group, which incorporated in HAp lattice as a structural defect
[39]. The difference between the spectra of HAp and Zn-HAp was a peak
at about 470 cm−1, indicating the ZnO band. FTIR spectra showed that Fig. 3. Chemical characterization of Zn-Ga:HAp NPs: A) FTIR and B) Raman spectra
after adding Zn2+ and Ga3+ in the HAp structure, the peaks became of HAp, Zn:HAp and Zn-Ga:HAp NPs.
wider, implying a decrease in the crystallinity and consequently structural
irregularities of nanopowders. In addition, the carbonate peak located at
890 and 1432 cm−1 in the spectrum of HAp was slightly reduced and wid- 3.3. Ions released in culture media
ened after ion doping. These peaks disappeared in the spectrum of HAp
doped with XGa = 0.2 and 0.6. According to research [39], this might be re- To investigate the functions of ionic release (PO3− 2
4 , Ca +, Zn
2+
, PO3−
4
3+
lated to Zn2+ and Ga3+ substitution with the carbonate group in pure HAp. and Ga ) on the degradation, antibacterial properties and cell behavior,
In addition, the intensity of phosphate peaks in the range of 400 to the release profiles of ions were determined in PBS for 7 days via ICP tech-
600 cm−1 significantly decreased compared to pure HAp, which could nique (Fig. 4). Our results showed that throughout the experiment, the ion
be attributed to the reduction of phosphate bonds or disruption of concentration was frequently decreased. In addition, the Ca2+ and PO3− 4
these bonds by co-doping Zn2+ and Ga 3+ in the structure of pure release profiles from the pure HA were low as compared to mono and
HAp, since the substitution of Ga3+ and Zn2 ions at Ca2+ sites were as- binary-doped HA nanopowders at different soaking times (Fig. 4A and B).
sociated with PO3− 4 and OH− ions. Decreased phosphate bonds were It could be related to the higher crystallinity of pure HAp than doped
also observed in other studies [37]. In addition, FTIR spectra of all ones (Table 1). In addition, according to the XRD patterns (Fig. 1A), β-
nanopowders doped with Zn 2+ and Ga 3+ , the OH band peak at TCP phase with high degradation rate was formed after ion doping leading
633 cm−1 showed a large decrease. It was also clear that the intensity to enhanced ion release rate. Similar result was reported in previous studies
of OH band in the range of 3568 cm−1 in doped nanopowders became [53]. In addition, during 7 days of incubation, the Ca2+ and PO3− 4 ion re-
weak. These results indicated that the removal of OH− groups could lease from Zn:HAp nanopowder was significantly higher than from Ga:
be due to the exchange of Ga3+ with Ca2+ in the apatite lattice. More- HAp nanopowders. Moreover, the concentration of Ca2+ released from
over, non-isovalent ionic substitutions could create Ca 2+ vacancies, Zn-Ga:HAp (Xzn = 0.1, XGa = 0.6) nanopowder was significantly lower
which might be electrically balanced by removing OH− groups [2]. than other Zn-Ga:HAp nanopowders. Generally, the dissolution rate of
The Raman spectra of the powders (Fig. 3B) also consisted of the peaks HAp in the buffer solution is a function of their crystallinity and the rate
at 598, 430 cm−1 related to the bending state of the O-P-O bond in phos- of dissolution increases with a decrease in their crystallinity [54]. High in-
phate. Moreover, the peaks at 1078, 1048 cm−1 demonstrated the asym- clusion of Ga3+ in the Zn:HAp structure enhanced the crystallinity of HAp
metric tension of PO bond in phosphate. The bond at 963 cm−1 was also nanopowder (Table 1), which prevented fast degradation leading to re-
attributed to asymmetrical tension of the PO bond in the phosphate. PO duced release of Ca2+and PO3− 4 ions in co-doped nanopowders.
bands were also observed in all samples at 430, 598 and 963 cm−1. After For Zn2+ and Ga3+ (Fig. 4C and D), there was a significant difference in
incorporation of Ga3+ and Zn2 ions and with increasing Ga content, the the ion release profiles among the different concentrated Zn2+ and Ga3+,
PO peak increased to 1048 cm−1. It could be concluded that Ga3+ and co-doped and single-doped HA. The Zn2+ release was the lowest from the
Zn2 ions were substituted at Ca2+sites whose bond position was Zn-Ga:HAp (XZn = 0.1, XGa = 0.6), while the Ga3+ release was the lowest
surrounded by PO3− 4 and OH [52]. from Zn-Ga:HAp (XZn = 0.1, XGa = 0.2). In addition, the Zn2+ and Ga3+

7
M. Shokri et al.
Fig. 4. The concentration of A) Ca2+, B) PO3−
4 , C) Zn
2+
and D) Ga3+ ions released from Zn-Ga:HAp, Zn:HAp and Zn-Ga:HAp NPs during 7 days soaking in PBS.
release was the highest from the mono-doped Zn-HA NPs and Ga:HAp (XGa
= 0.6), respectively. Higher zinc ion release might be attributed to the
smaller particles sizes of Zn: HAp nanopowder leading to higher specific
surface area and its higher crystallinity degree. In addition, our results dem-
onstrated that that binary-doping significantly reduced the Zn2+ and Ga3+
ion release. It could be demonstrated that Ga3+ reduced the release of other
ions in HAp powder. The role of Ga3+ to promote the stability and decrease
the degradation rate of various formulation was also similarly confirmed,
before [55]. Sahdev et al. [56] found that an increase of gallium in bioac-
tive glass led to a decrease in the release of other ions as well as overall
dissolution. However, in Zn-Ga:HAp (XZn = 0.1, XGa = 0.4) nanopowder,
all sites were filled with Ga elements and, hence, the degradation rate
and release of Zn2+ ions increased compared to other Zn-Ga:HAp
nanopowders. In addition, the concentration of Ga3+ released in the Ga:
HAp nanopowders enhanced with increasing amount of Ga doping in the
HAp structure. The same trend was detected for Zn-Ga:HAp nanopowder.
This could be related to the presence of more Ga3+ions doping in the HAp.
3.4. In vitro antibacterial activity of Zn-Ga:HAp nanopowder
Antibacterial activity of HAp nanopowders binary-doped with gallium
and zinc (Zn:HAp, Ga:HAp, Zn-Ga:HAp) was examined against two kinds
of bacteria, S. aureus and E. coli. The viability of bacteria preserved with dif-
ferent concentrations of nanopowders (125, 250, 500 and 1000 μg/ml) is
presented in Fig. 5A, B. According to the results, the level of antimicrobial
activity against both E. coli and S. aureus bacteria in all doped HAp
nanopowders was significantly higher than pure HAp nanopowder. Zn:
8
Biomaterials Advances 134 (2022) 112684
HAp NPs also supposed to well inhibit bacterial adhesion and growth via re-
leasing zinc ions and generating reactive oxygen species (ROS) from ZnO.
The bacterial mortality of both strains in Zn-Ga:HAp nanopowders signifi-
cantly increased compared to Zn:HAp nanopowders and comparable with
Ga:HAp nanopowder. In another word, higher gallium concentration
showed more significant antimicrobial activity. With increasing gallium
concentration in Zn-Ga:HAp nanopowders from XGa = 0.2 to XGa = 0.6,
the survival rate of bacteria decreased from 40% to about 20%. Survival
of S. aureus bacteria in Ga:HAp nanopowders)XGa = 0.2 (reduced to less
than 20%. According to Fig. 4, the release of Ga ions enhanced with increas-
ing the gallium concentration in both Ga:HAp, Zn-Ga:HAp nanopowders.
Therefore, improved antibacterial activity of Ga:HAp and Zn-Ga:HAp
nanopowders with increasing gallium content could be attributed to the en-
hanced synergic role of gallium and zinc ion release. According to previous
studies, gallium can interrupt bacterial growth via improved ROS produc-
tion in cells due to the generation of Ga3+ ions penetration and intracellular
diffusion, which hinders bacterial division and proliferation [57]. In addi-
tion, antibacterial activity of gallium is intensely related to the inhibition
of Fe-dependent pathways owing to its ability to compete for the iron-
binding sites uptake (due to the high similarity of these two ions, including
similar ion radius, electronegativity, neighbourhood number, etc.) [58].
The findings of the current study in terms of antibacterial activity were con-
sistent with those reported by Albulym et al. [48], who observed that
higher concentrations of Ga3+ ions had higher antimicrobial capacity.
They also stated that S. aureus was susceptible to Ga3+ ions, which showed
to be highly sensitive in this study. In a study, Kurtjak and colleagues [31]
demonstrated the antibacterial action of Ga3+-doped HAp against E. coli.
M. Shokri et al.
Fig. 5. The antibacterial activity of Zn-Ga:HAp nanoparticles: The percent viability of A) E. coli and B) S. aureus treated with different concentrations of Zn-Ga:HAp NPs
compared to Zn:HAp, Ga:HAp and HAp). Representative SEM images of C) E. coli and D) S. aureus treated with Zn-Ga:HAp nanopowders compared to Zn:HAp, Ga:HAp
and HAp.
According to our results, the presence of gallium ions greatly enhanced the
antibacterial properties of Zn:HAp nanopowder and reduced cell viability
from 48% to 6% in S. aureus and from 57% to 18% in E. coli.
Compared to the survival results of E. coli and S. aureus, it was clear that
the E. coli strain was more resistant than S. aureus in all doped HAp
nanopowders. The dissimilar results between Gram-positive and Gram-
negative bacteria strains could be attributed to diverse bacterial properties
and morphology, including the presence of a thin layer of peptidoglycan on
Gram-negative bacteria compared to layer free Gram-positive bacteria. This
layer makes gram-negative bacteria such as E. coli more resistant to antimi-
crobial agents than gram-positive bacteria such as S. aureus [59]. In addi-
tion to the ion doping and bacteria strains, the concentration of
nanopowders could also significantly modulate the bactericidal activity.
As can be seen, with increasing the concentration of doped nanopowders
in bacterial culture medium, the survival rate was significantly decreased.
Moreover, in Zn-Ga:HAp NPs, the slope reduced to 500 μg/ml and then
the slope significantly reduced. It could be concluded that, the MIC for
both bacterial strains was 500 μg/ml. However, in Ga:HAp nanopowders
at a concentration of 250, a constant amount of mortality was obtained,
which could be attributed to the greater effect of gallium ion on bacterial
metabolism. Therefore, in Ga:HAp nanopowders, the minimum inhibitory
concentration could be reported as 250 μg/ml.
Adhesion and proliferation of E. coli and S. aureus microbial cells were
examined after 24 h of incubation in a suspension containing pure HAp
and Zn:HAp and Zn-Ga:HAp nanopowders using SEM imaging. According
to Fig. 5C, D, the bacteria in the control sample adhered to the surface
and accumulated. The morphology of both bacteria remained intact and
covered the surface of the glass slide. Conversely, stabilized bacteria in
doped HAp nanopowders were few and scattered, and bacterial debris
9
Biomaterials Advances 134 (2022) 112684
was observed in Zn-Ga:HAp nanopowders. SEM images of E. coli (Fig. 5C)
in contact with pure HAp nanopowders were slightly affected and showed
little inhibitory activity compared to the control sample. While Zn:HAp
nanopowders had more antibacterial activity than HAp due to ROS produc-
tion, significant and more vigorous antibacterial activity was observed for
Zn-Ga: HAp nanopowders. As the concentration of gallium in Zn:HAp
nanopowders increased, the accumulation and number of bacteria on the
surface decreased and had an inhibitory effect on bacterial cell growth.
Also, a change in the bacteria morphology was observed in contact with
Zn-Ga:HAp nanopowders, emphasizing the reaction of nanopowder with
the membrane components and cell wall of bacteria. It can lead to irrevers-
ible changes in their structure, bacterial cell death [60]. In the case of
S. aureus microbial suspensions incubated with NPs, the bacterial cells pre-
sented in the round-shape with the size of 0.8–1.2 μm. SEM images
(Fig. 5D) showed a robust inhibitory effect on the adhesion and growth of
S. aureus cells in contact with HAp nanopowders. By co-doping Zn2+ and
Ga3+ ions into the HAp structure, a significant change in the morphology
of bacteria was detected. Doping ions caused ions to accumulate in the
cell membrane of bacteria, resulting in a difference in their permeability
(gradual release of proteins and lipopolysaccharides). This prevented the
transfer of protons through the cell membrane, resulting in cell membrane
destruction and bacterial cell death [61].
3.5. In vitro cell interaction with Zn-Ga:HAp nanopowder
The cytotoxicity of Zn-Ga:HAp NPs was assessed using MTT assay after
24 h and 72 h. According to Fig. S2, it was demonstrated that the Zn2+ sub-
stitution in HAp structure enhanced the proliferation of hMSCs compared
to HA. It could be related to the important role of Zn species in biological
M. Shokri et al.
functions, such as DNA synthesis, bone formation and biomineralization.
However, a higher concentration of Zn2+ ions (XZn > 0.2) significantly de-
creased the hMSCs growth rate. To improve the concentration limit of Zn2+
without any adverse effect, Ga3+ was binarily doped with Zn2+ to balance
the cytotoxic effects of Zn2+ and encourage the proliferation and differen-
tiation of hMSCs. Here, the viability of hMSCs was tested in the presence of
pure HAp, Ga:HAp (XGa = 0.2, 0.4 and 0.6) and Zn-Ga:HAp (XZn = 0.1 and
XGa = 0.2, 0.4 and 0.6) nanopowders in three different concentrations
(250, 500 and 1000 μg/ml). According to Fig. 6, the cell viability was
higher than 80% after 24 h and 72 h, indicating no toxic effect on the
cells. However, after 24 h incubation (Fig. 6A), at higher concentrations
of nanopowders, HAp NPs were likely to agglomerate and precipitate on
cells, causing cell death. By increasing the concentration of Ga in HAp
nanopowders from XGa = 0.2 to XGa = 0.6, the cell viability decreased
compared to HAp. Similarly, in Zn-Ga:HAp nanopowders with XGa = 0.6,
the lowest cell viability was achieved after 24 h and 72 h (85 and 78%, re-
spectively). Therefore, it could be concluded that increasing Ga3+ ions to
some extent led to a decrease in cell proliferation and very high concentra-
tions may lead to cytotoxicity. Rana et al. [4] also showed that a steady but
significant decrease in cell viability with increasing gallium content. How-
ever, they showed no cytotoxicity effect on cells. Gallium has been reported
to increase cell membrane permeability, thereby destabilizing electrical
charges at the cell surface. Consequently, it increased the flow of calcium
from the mitochondria, initiated the early stages of apoptosis, and may re-
duce cell viability. Moreover, it has been reported that Ga3+ shares similar-
ities with Fe3+ and it may bond with iron-binding proteins. Consequently,
gallium could interrupt protein functions and result in negative down-
stream effects on cells [62]. Therefore, in Ga:HAp (XGa = 0.6) and Zn-Ga:
HAp (XZn = 0.1 and XGa = 0.6), the high amount of Ga3+ ions could
begin negative effects on hMSCs.
The synergic role of Zn/Ga ion on the osteogenic behavior if hMSCs is
another crucial goal of this study. The ALP produced by osteoblast cells, is
used as a parameter to express the activity of osteoblast cells [63,64]. It
needs to mention that ALP must be increased to ossify and observe the dif-
ferentiation of MSCs into bone cells [63]. In this study, the amount of ALP
enzyme expressed in contact with samples at three time points of 3, 7 and
14 days was studied and compared to the control. The quantitative analysis
of the ALP activity for all nanopowders is shown in Fig. 7A. Our results re-
vealed that co-doping Zn2 + and Ga3 + ions in HAp nanopowders modu-
lated their osteogenic potential. After 3th, 7th and 14th day of cell
culture, the ALP activity for Zn:HAp, Ga:HAp and Zn-Ga:HAp nanopowders
Fig. 6. Cytotoxicity of Zn-Ga:HAp nanoparticles on hMSCs: MTT assay for Ga-Zn:HAp NPs with different concentrations on hMSCs after A) 24 h and B) 72 h (*: P < 0.05).
10
Biomaterials Advances 134 (2022) 112684
enhanced compared to control and pure HAp nanopowders. The ALP activ-
ity of all doped nanopowders increased with incubation time, indicating
good differentiation of hMSCs in Zn-Ga:HAp nanopowders. Noticeably,
HAp nanopowders doped with Ga3+ (XGa = 0.2) on days 3, 7 and 14
showed significantly (P < 0.05) increased activity compared to pure HAp.
However, the ossification effect of Ga3+ was more pronounced at lower
concentrations, as ALP activity decreased with increasing gallium release
from XGa = 0.2 to XGa = 0.6. Similarly, Yu el al. [65] found that the ALP
activity of osteoblasts enhanced with increasing gallium concentrations,
reaching a maximum level of ALP activity at 10−4 M. In addition, at gallium
concentrations above 10−4 M, ALP activity decreased. Their result demon-
strated that Ga ions could affect osteoblast ALP activity in a concentration-
dependent manner. Based on our results, XGa = 0.2 was selected as the
optimized sample that induced the highest levels of ALP activity.
In addition, the presence of zinc also supported the cell differentiation
and showed high expression of ALP in contact with Zn:HAp NPs [64,66,
67]. Zn2 + doping alone (XZn = 0.1) increased the ALP activity in the
HAp structure, indicating the ability of zinc ions in ossification. While the
mechanism underlying enhanced bone formation of zinc ions is unclear,
zinc at concentrations several times higher than the physiologic level
could result in an increase in ALP activity of osteoblasts [68]. Our findings
are in agreement with the finding of Ito et al. [69], who showed that bone
marrow stromal cells (BMSc) cultured on zinc-containing calcium phos-
phates differentiated into osteoblast-like cells more than cells on zinc-free
ceramics. With co-doping Zn2+ and Ga3+ ions into the HAp structure, the
amount of ALP activity in Zn-Ga: HAp nanopowders significantly increased
compared to the pure HAp due to the simultaneous effect of two ions.
Higher ALP activity was observed for Zn-Ga:HAp (XZn = 0.1 and XGa =
0.4) on days 3 and 7, which might be related to higher release of zinc
ions during the culture time. However, with a further increase in Ga3+ con-
tent to XGa = 0.6, the activity decreased and no significant difference was
observed after day 14. It could be related to decreased release of zinc ions
and enhanced release of gallium ions which postpone the differentiation
of hMSCs. So, the presence of Zn2+ and Ga3+ ions increased the ALP activ-
ity and further stimulated the differentiation of hMSCs into osteoblasts. The
consequences of ALP indicated that the differentiation of osteoblasts in-
creased with trace elements doping from HAp to Zn-Ga:HAp.
Alizarin red staining was performed to study the ability of the nanoparti-
cles to promote hMSCs mineralization after 14 days of cell culture. Fig. 7B, C
shows the result of alizarin red staining in contact with the samples. In view
of all of these findings, we speculate that Zn-Ga:HAp could trigger osteogenic
M. Shokri et al.
Fig. 7. Osteogenic activity of hMSCs in contact with Zn-Ga:HAp NPs: A) ALP activity after 3, 7 and 14 days of culture. B) Representative images and C) quantitative analysis of
Alizarin Red S staining of hMSCs cultured in basal medium for 14 days (*: P < 0.05).
differentiation of hMSCs and then stimulate the proliferation of their miner-
alizing osteoblast progeny. According to the quantitative analysis of mineral-
ization, co-doping of Zn2+ and Ga3+ ions resulted in a significant increase in
the mineralization compared to the pure HAp (P < 0.05). However, the pres-
ence of Ga3+ in the HAp structure showed less mineralization than Zn:HAp
nanopowders. In addition, at higher concentrations of gallium (XGa = 0.6),
a decrease in the mineralization was observed. These quantitative results
were also consistent with microscopic images of alizarin red staining
(Fig. 7B). In the control sample, hMSCs were not differentiated and did not
appear red after staining with alizarin. Lack of staining of the bottom of the
well was a sign of the absence of calcium on the surface and cell differentia-
tion did not occur. In contrary, Zn-Ga:HAp NPs (XZn = 0.1 and XGa = 0.4)
resulted in more intense red-orange colour than other wells, confirming
more calcium deposition and higher differentiation of hMSCs to osteoblast
cells. The mineralization of Zn-Ga:HAp NPs was comparable with Zn:HAp
NPs demonstrating the significant role of zinc ion release on hMSCs differen-
tiation and mineralization, in agreement with results of ALP activity. Judging
from all these findings, Zn-Ga:HAp NPs enhanced calcium nodule formation
in hMSCs compared with control cells and HAp. However, this co-treatment
with gallium and zinc revealed a concentration-dependent manner; calcium
nodule formation decreased with increasing gallium content.
4. Conclusion
In this study, Zn2+ and Ga3+-containing hydroxyapatite nanoparticles
(Zn-Ga:HAp NPs) were synthesized by simple sol-gel method and the synergic ✓ All authors have participated in (a) conception and design, or analysis and
effects of co-doping of Zn2+ and Ga3+ ions on the physicochemical properties
and biological activity of HAp structure were investigated. Co-doping of Zn2+
and Ga3+ ions resulted in the formation of secondary TCP, reduced crystallin-
ity and particle size of HAp leading to improved solubility and bioactivity. In
Biomaterials Advances 134 (2022) 112684
addition, Zn2+ and Ga3+ ions release from Zn-Ga:HAp NPs was significantly
changed, depending on the concentration of doped ions, which could signif-
icantly modulate the antibacterial activity and hMSCs functions. The antibac-
terial activity of HAp significantly improved against both S. aureus and E. coli
bacteria, after co-doping of Zn2+ and Ga3+. Substitution of Zn2+ and Ga3+
ions in the HAp structure not only improved its biocompatibility towards
hMSCs, but also supported the osteogenesis in basal medium. The novel Zn-
Ga:HAp NPs is a favorable candidate for bone tissue engineering and implant
coatings. Future work will be focused on more detailed in vitro and in vivo
studies, and on the development of Zn2+ and Ga3+ doped HAp carriers for
controlled drug release.
CRediT authorship contribution statement
M.S.: Investigation, Writing the original work, Visualization, Formal
analysis, Software
M.K.. Supervision, Project administration, Funding acquisition,
Conceptualization
H.A.T: Supervision, Funding acquisition
M.B.E: Validation, Resources
R.M.A: Validation, Resources
Declaration of competing interest
interpretation of the data; (b) drafting the article or revising it critically for
important intellectual content; and (c) approval of the final version.
✓ This manuscript has not been submitted to, nor is under review at, an-
other journal or other publishing venue.
11
M. Shokri et al.
✓ The authors have no affiliation with any organization with a direct or
indirect financial interest in the subject matter discussed in the
manuscript
✓ The following authors have affiliations with organizations with direct or
indirect financial interest in the subject matter discussed in the manu-
script: Mahshid Kharaziha
Acknowledgements
This study received financial support from the National Institute for
Medical Research Development (NIMAD), Islamic Republic of Iran (Grant
Number:984282).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.msec.2022.112684.
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