Journal of Non-Crystalline Solids 554 (2021) 120613

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Journal of Non-Crystalline Solids

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

Sol-gel derived silicate-based bioactive glass: Studies of synergetic effect of

zirconium and magnesium on structural and biological characteristics
Amirhossein Moghanian a, *, Mahzad Haji Mahdi Tajer a, Mohammadamin Zohourfazeli a,
Zahra Miri b, MortezaSaghafi Yazdi a
a
Department of Materials Engineering, Imam Khomeini International University, Qazvin 34149-16818, Iran
b
Department of Materials Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

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

Keywords: The 5 mol.% zirconium-incorporated 58S bioactive glass (Zr-BG) was modified by addition of magnesium (Mg).
In vitro Bioactivity These BGs were synthesizedby substitution of CaO with MgO (0, 1, 3, 5 and 7 mol.%) through the sol-gel method.
Sol-gel processes The SEM, FTIR and XRD were confirmed the formation of hydroxyapatite (HA) on BGs surfaces, after 7th day of
ZrO2
immersion in simulated body fluid (SBF) solution. The results of MTT assay and alkaline phosphatase (ALP)
MgO
activity analyses showed that the specimen with 5 Zr and 5 Mg (mol.%) in composition stimulated the cell
Bone tissue engineering
proliferation and ALP activity more than the other specimens. Mechanical properties was proved by Vickers
hardness test. The ZBG-M3 and ZBG-M5 were introduced as the reliable BGs due to the best synergetic effect of
both ions through in vitro evaluations and candidates for further in vivo experiments.

1. Introduction advantages such as capability of performing at room temperature [23],

Various types of injuries or burnouts lead to hard and soft tissue
defects, which need treatment and healing [1–3]. The initial clinical
biomaterials were bio-inert and biocompatible [4], but then with the
completion of research, bioactive property was obtained [5]. It helped a
lot in accelerating recoveryprocess by mechanism of making connec­
tions with host tissue [6,7]. This capability was due to formation of a
layer of calcium-phosphate based ceramic (Ca10(PO4)6(OH)2), which
was introduced as hydroxyapatite (HA), on the surface of bioactive
glasses (BGs) [8]. This claim was first made in the studies of Hench et al.
[9] after research on a substance in 45% SiO2-24.5% Na2O-24.5%
CaO-6% P2O5 quaternary system.
For further development of composite system [10–12] and obtaining
bi-functional implants, biomaterials were manufactured by combination
of bioactive composites [13] and BGs [14,15], as scaffolds for tissue
engineering [16,17] and personalized medicines [18,19]. These mate­
rials were made through different methods such as sol-gel [20],
melt-quenching [21], and so on. Although specific characteristics of
each route affect the quality of the final product, other important factors
such as economic efficiency and being eco-friendly are considered for
choosing the manufacturing method [22]. Among these various pro­
cedures, the sol-gel method is of interest to researchers, due to its
higher purity, greater bioactivity of the final material, and ease of the
process [24].
Over the last few years, all efforts have been made to find novel BGs
with more applications and generally improve the quality of BGs. This
modification was achieved by applying changes in chemical composi­
tion of BG [25–27], numerical and simulation studies [28], etc. These
multifunctional designed BGs were used in various clinical usages such
as angiogenesis [29], drug delivery [30], light-triggered [31,32] sys­
tems, and so on.
Presence of magnesium (Mg) in bone, enamel, and muscle structures,
have encouraged researchers to use Mg-based or containing biomaterials
in the human body [33,34]. Pure Mg, with an elastic modulus close to
the bone, provided a wide range of applications for scientists and sur­
geons [35]. Gradually, after the invention of BGs, research began on the
properties of Mg-modified BGs (Mg-BGs) and the effect of different
amounts of Mg on the performance of BGs [36,37]. The key role of this
modification was ability of Mg to improve the mechanical properties
[38] as well as enhancing formation rate of in vitro and in vivo HA par­
ticles [39]. It has been reported that function of Mg in BG is affected by
(1) molar percentage of Mg and (2) presence of other elements in the
composition [33,40].
By 2012 [33], summarizing the results of research on the optimum

* Corresponding author.
E-mail address: moghanian@eng.ikiu.ac.ir (A. Moghanian).

https://doi.org/10.1016/j.jnoncrysol.2020.120613
Received 5 October 2020; Received in revised form 27 November 2020; Accepted 14 December 2020
Available online 5 January 2021
0022-3093/© 2020 Elsevier B.V. All rights reserved.
A. Moghanian et al.
Table 1
The designed chemical composition and label of specimens (mol%).
Sample SiO2 CaO P2O5 ZrO2 MgO
ZBG-M0 mol% 60 31 4 5 0
ZBG-M1 mol% 60 30 4 5 1 2.1. Materials and BG’s synthesis
ZBG-M3 mol% 60 28 4 5 3
ZBG-M5 mol% 60 26 4 5 5 The preparation of BGs were done by utilizing tetraethyl orthosili­
ZBG-M7 mol% 60 24 4 5 7 cate (TEOS), calcium nitrate tetrahydrateCa(NO3)2.4H2O, triethyl
amount of Mg in the structure of BG showed that a reliable applicable
range of Mg content was not selected, yet. But in 2013, Prabhu et al.
[41], reported that incorporation of Mg over 10 mol.% was inefficient
and weakened the biological properties of BG. In the following, in­
vestigations on contents of Mg less than 10 mol.% (1, 3, 5, and 10) were
done and the optimum amount of Mg-incorporation was introduced as 5
mol.%, by Tabia et al [42]. Moreover, Moghanian et al. [39] evaluated
the effect of Mg substitution with Ca (1, 3, 5, 8, 10 mol.%) on structural
and biological properties of this modified 58S-BG. They reported the
statistically significant improving performance of 5 (mol.%) addition of
Mg in comparison to other contents. They also stated that specimens
with 8 and 10 (mol.%) of Mg revealed the less stimulating effect on cell
viability and proliferation toward 5 (mol.%) Mg-containing specimen.
The history of research on biomaterials showed that zirconium (Zr)
was first frequently used as the main composition of bio-inert implants
due to its good mechanical properties [43,44]. Furthermore, it has been
reported that the incorporation of Zr in BGs composition improved
biological properties of BGs through in vitro evaluations [45–47].
Addition of Zrdensifies the structure of the BG, which is because of its
higher electronegativity and greater tendency to form stronger covalent
bonds in comparison to Ca-O bonds [46]. This had a good effect on
mechanical properties of BG. It should be noted that in a previous
investigation on Zr-incorporated 58S silicate-based BG (Zr-BG) [46], the
best amount of Zr addition was considered 5 (mol%) in the whole
composition.
In this study, the effect of adding different amounts of Mg on the
performance of Zr-BG with an optimum percentage of Zr (5mol.%) has
been evaluated. These BGs were designed in the 60% SiO2-(36-X)% CaO-
4% P2O5-5% ZrO2- X% MgO (which X=0, 1, 3, 5 and 7 mol.%) system
and then successfully synthesized.The main purpose of this research was
to study the effects of simultaneous presence of Zr and Mg on structural,
mechanical, and biological properties of 58S BG. These investigations on
the section of structural analysis were done by the means of scanning
electron microscopy (SEM), X-ray diffraction (XRD) and Fourier trans­
form infrared spectrum (FTIR)analysis. The effect of the synthesized BGs
on function of MC3T3 cells was investigated through analyses such as 3-
(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)
assay and alkaline phosphatase (ALP) activity. Moreover, antibacterial
performance of BGs was studied by culturing them with methicillin-
resistant Staphylococcus aureus (MRSA). The accuracy of the obtained
results was checked out by applying cell culturing techniques. After the
in vitro biological evaluations in simulated body fluid (SBF) solution,
changes of SBF solution before and after the immersion of BGs were
investigated by applying inductively coupled plasma atomic emission
spectroscopy (ICP-AES) analysis and pH measurement. To provide a
complete research on the properties of these BGs, their mechanical
properties were also evaluated by Vickers hardness assay. Another goal
of this scientific work is to try to obtain new improved BGs that have
versatile capabilities for tissue engineering and drug delivery as clinical
Table 2
The components of human blood Plasma and the SBF solution (mmol.L− 1).
Ion Na+ K+ Mg+2
Plasma (mmol.L )–1
142.0 5.0 1.5
SBF (mmol.L–1) 142.0 5.0 1.5
2
Journal of Non-Crystalline Solids 554 (2021) 120613
applications.
2. Material and methods
phosphate (TEP), zirconium (iv) propoxide(C12H28O4Zr), and magne­
sium nitrate hexahydrate Mg(NO3)2.6H2O as the precursors of SiO2,
CaO, P2O5, ZrO2 and MgO in sol-gel derived BG, respectively. It should
be noted these precursors were obtained from Sigma-Aldrich. These
materials were used without further purification based on stoichio­
metric amounts related to the chemical composition of BGs (Table 1).
Moreover, deionized water and nitric acid were used to make 2 molar
acid solution in order to dissolve TEOS in it, as the first step of synthe­
sizing. In the next stages, each time a transparent solution was observed,
following materials were added in order of TEP, calcium nitrate tetra­
hydrateCa(NO3)2.4H2O, zirconium (iv) propoxide, and magnesium ni­
trate hexahydrate Mg(NO3)2.6H2O. The final powder was obtained after
7 days of natural (room temperature, 25 ◦ C), 3 days of artificial (oven,
75 ◦ C) aging, and 3 h of calcination (furnace, 700 ◦ C). These heat
treatment cycles were performed to remove the remaining nitrates and
water as well as condense the silanol groups. Afterwards, grounded
powders were shaped as discs with Ø10 × 3 mm. The weight of each disc
was equal to 0.32 mg.
Eventually, the preparationof simulated body fluid (SBF) solution
was done for in vitro biological analysis (based on the procedure of
Kokubo [48]). Utilizing SBF was because of the fact that its composition
is in high similarity with human blood plasma, as Kukobu [49] has
introduced (Table 2). Each disc was immersed in 6.50 ml of SBF solution
(m/V= 0.32 mg/ 6.50 ml).
2.2. Thermal analysis
The synthesized BGs were evaluated by the differential thermal
(DTA) and thermogravimetric (TGA) analysis in order to select the
suitable temperature for sintering. A Shimadzu TGA-50 was utilized for
recording thermograms (DTA/TGA) in heat range of 25–1200 ±5 ◦ C and
under the protection of N2 as an inert gas (60 mL.min–1 and constant
flow)
2.3. Characterization of BG surface and SBF solution
FTIR (FTIR, Nicolet Avatar 660 (Nicolet, USA)), SEM (SEM, Philips
XL30, Netherland) and XRD (XRD, INEL-Equinox-3000, France) analysis
were used to detect the formation or non-formation of the HA layer over
the surface of BGs. Sample preparation steps for the FTIR test started by
mixing the powders with 100 mg KBr (spectroscopy grade) and then
continued by pelletizing and vacuuming the mixture. After the samples
were prepared, FTIR analysis was performed with a resolution of 8 cm− 1
(λ = 400 – 4000 cm− 1) Samples were prepared for SEM analysis (V=10
Kv)by applying one layer of gold as the coating. The surface of the BGs
was examined by XRD analysis by irradiating CuKα radiation with a
wavelength of 0.15406 nm(40 kV). Moreover, the X-ray spectroscopic
analysis system (EDS) analysis was done and the chemical composition
of the surface after immersion of BGs in SBF was checked.
Complementary analysis was performed on the SBF solution by
examining the difference between the pH value of the solution and the
Ca+2 Cl– HCO–1
3 HPO–2
4 SO–2
4
2.5 103.0 27.0 1.0 0.5
2.5 147.8 4.2 1.0 0.5
A. Moghanian et al. Journal of Non-Crystalline Solids 554 (2021) 120613

concentration of ions before and after immersion of the BGs in the so­
lution. The pH values were measured by the mean of a calibrated pH
meter (Corning 340, USA) as well as applying ICP-AES (Varian Vista Pro,
Palo Alto, USA) analysis for evaluating ion concentrations.

2.4. Mechanical evaluations

The changes in mechanical properties that had occurred after im­

mersion of the BGs in the SBF solution, was examined. This was moni­
tored by utilizing the Vickers hardness (BulutMakina HVS 1000,
Turkey). To do so, 10 indents were applied by loading 100 gf (Max) for
10 s.

2.5. Biological evaluation

2.5.1. MTT assay

The first biological assay was the measurement of optical density
(OD) by the mean of a multi-well microplate reader (λ = 570 nm, EL
312e Biokinetics reader, Biotek Instruments) through MTT analysis.
After evaluating the OD of the control sample (A) and the selected
sample (B), the percentage of cell viability was achieved, as follows
[50]:
%Cellviability = B/A × 100 (1)
3
For this aim, cells were co-cultured by density of 6 × 10 cells per
well along with synthesized BGs and culture medium. After incubation
of the plate for 4 h at 37 ◦ C, the culture medium was gently pipetted out
for the addition of the dimethyl sulfoxide (DMSO, Sigma), as the next Fig. 1. The DTA/TGA curves of ZBG-M0 and ZBG-M7.
step. This was applied by the aim of dissolving the precipitated for­
mazan. In the end, was used to measure OD.

2.5.2. Alkaline phosphatase (ALP) activity

The amount of conversion of p-nitrophenyl phosphate to p-nitro­
phenol was measured in order to investigate the ALP activity of MC3T3-
E1 osteoblast-like cell line. The procedure was the same as the method,
Elgendy introduced [51]. According to that, the preparation of speci­
mens was started by culturing the cells (density of 1 × 104 cells.cm–2)
with synthesized BGs at 37 ◦ C with and providing a humidified atmo­
sphere. In the following, supernatant fluid was removed before rinsing
the cell layer with PBS solution. This removal was done by the mean of a
pipette. Afterward, 1 ml Tris buffer was used for homogenizing the cells.
After the sonication of cells on ice (4 min), the equal volumes of 20 μL of
1ml of a p-nitrophenyl phosphate solution (pNPP, Sigma, 16 mmol/L)
was added and then, the plate was incubated at 30 ◦ C (5 min).

2.5.3. Antibacterial studies

MRSA was selected to perform antibacterial studies against it. MRSA
is a type of Staphylococcus aureus infection that is known as the bacteria
with the capability of exhibiting resistance toward some antibiotics and
causing infections after clinical applications [52]. Research on the
bactericidal function of BGs was done by culturing the MRSA bacteria
with BGs and cells at 37 ◦ C [53]. The colony-forming units per milliliter Fig. 2. The FTIR spectra of (A) all samples after 7 days of soaking and (B) ZBG-
(CFU/ml) [54] method was utilized, in order to count the number of M5 in various soaking times in SBF solution.
viable (X) and entire bacteria (Y). Eventually, the last step was calcu­
lating the antibacterial fraction, as follows [54]: [55].
Antibacterialfraction = 1 − (X / Y). (2)
2.6. Statistical analysis
2.5.4. Cell staining techniques
Statistical comparisons were done overall specimens (at least 5 in­
Two techniques of cell staining were selected to confirm the accuracy
dependent experiments) and data were declared by the form of mean ±
of the obtained results. On one hand, the Live/Dead cell staining is
standard deviation (SD) and also, probability value (p) (*p <0.05 sta­
focused on showing the live and cells after co-culturing with synthesized
tistically significant). The analysis of data in terms of statistical signifi­
BGs. And on the other, Dapi/Actin cell staining signifies the effect of the
cance was performed by utilizing GraphPad Prism (V.3.0, GraphPad
mentioned co-culturing on nuclei and cytoskeleton of MC3T3-E1 oste­
Software, USA).
oblast-like cells. The applied procedure of these cell staining methods
were completely similar to what was introduced in the previous article

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A. Moghanian et al. Journal of Non-Crystalline Solids 554 (2021) 120613

Table 3 1094, and 1250 cm− 1). But on the 7th day, new peaks appeared at
The wavelengths of chemical groups presentend in FTIR spectra of synthezide wavelengths of 570 and 603-605 assigned to the P–O stretching vibra­
BGs. tional bonds bands. Observation of these peaks along with Si related
Wavenumber (cm− 1) Group peaks, indicated the formation of a HA layer on the surface of synthe­
470 Si-O-Si
sized BGs. This is mentioned as the 4th stage from 5 sequential steps of
804 Symmetric Si-O-Si stretching in SiO4 tetrahedron HA formation, by Pereira et al. [56] The same values for Si related bonds
1094 Si-O-Si symmetric stretching were reported from Zr and Mg containing BGs [41,45]. The increasing
1250 Si-O-Si asymmetric stretching intensity of peaks related to P-O bonds in the 14th day of immersion in
570 V2 – PO3− 4
comparison to the 7th day, revealed the fact that as the immersion time
603-605 V3 – PO3− 4
1450 V2 – CO2− 3 elongated, the more HA particles covered the surface of BGs. Moreover,
it can be claimed as the same for less peak intensity of P-O bonds in
specimens with higher 5 (mol.%) Mg content in the composition, due to
3. Results and discussions the less density of the HA layer on the surface. Meanwhile, the
mentioned process can be proved by SEM images.
3.1. Thermal analysis
3.3. SEM images
The presented DTA and TGA thermograms are dedicated to the ZMG-
M0 and ZMG-M7, as shown in Fig. 1. The main 3 stages of weight loss of Fig. 3 depicts the captured images from the surface of synthesized
BGs that occurred through the performing of heat treatment was BGs before and after immersion in the SBF solution. The first thing to
observed in TGA thermograms. These stages were assigned to volatili­ notice was that the rough surface, seen in the pre-immersion images,
zation of remaining water, condensation of residual silanols and was changed to a surface covered by spherical masses. Another point to
removal of the nitrate groups. The percentage of weight reduction of consider was that the quantity of this new layer was increased over time.
ZBG-M7 was more than ZBG-M0 according to the more water and nitrate The formation of this new layer over the surface after the 7th day of
of Mg containing sample because of magnesium nitrate hexahydrate, as immersion was in accordance with the results of FTIR analysis. There­
the precursor of Mg. fore, this layer of globular deposits can be introduced as the HA layer.
The previously reported investigation revealed that the formation of the
HA layer on the surface of Zr-BGs occurred after 7 days of immersion in
3.2. FTIR analysis the SBF solution [46]. According to the obtained results, it can be stated
that the incorporation of Mg contents did not have a retarding effect on
The FTIR spectra of synthesized BGs is selected in a range of 400 to the formation of the HA layer. This was also in agreement with the
2000 cm− 1 and shown in Fig. 2(A, B). The wavelength related to each monitored chemical composition of BGs.
structural group is gathered in Table 3. According to Fig. 1 (A), the FTIR
spectra of specimens exhibited the characteristic peaks related to
Si–O–Si bonds before and after immersion until the 3rd day (470, 804,

Fig. 3. SEM images of ZBG-M3 and ZBG-M5 before, after 7 and 14 days of immersion in SBF solution and EDS of both samples after 7 days.

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A. Moghanian et al. Journal of Non-Crystalline Solids 554 (2021) 120613

Fig. 4. XRD patterns of (A) all samples after 7 days of immersion and (B) the
ZBG-M5 in different periods of immersion in SBF solution.

3.4. XRD phase analysis

Fig. 4 indicates the obtained XRD patterns of synthesized BGs (20◦

<2θ< 60◦ ). The standard JCPDS cards(no. 09-0432) has introduced the
angles related to HA structure at 2θ= 25.8◦ , 31.9◦ ,and 48. As demon­
strated in Fig. 4(A), peaks were not observed at the mentioned angles
over 1st and 3rd day of immersion; but after 7 days of immersion, these
peaks became apparent. Similarly, the appearance of these peaks in the
XRD patterns of all samples after 7 days of immersion, indicated and
confirmed the formation of HA on the surface of all samples. It should be
noted that these peaks were related to 3 crystalline planes of(002),
(211), and (222), respectively. Previously, the same peaks were reported
about Zr or Mg incorporated BGs [42,46]. Moghanian et al. [57] and
Tabia et al. [42] introduced that the formation of HA particles was
retarded for silicate-based BGs until the 7th day of immersion in SBF. The
same retarding effect was observed in our recent paper for Zr-containing
BG [46], which the HA layer formed after 7 days. In this article, it can be
concluded that the simultaneous presence of Zr and Mg in structure did
notdelay the formation of primary HA particles. It is obvious that the
intensity of peaks of ZBG-M5 and ZBG-M7 gradually decreased as the
amount of Mg incorporation increased over 5 (mol.%). Based on the
results of these analyses from the surface of BGs, it could be claimed that
ZBG-M5 had the most acceptable ability to form more HA than other
samples.

3.5. ICP assay

Fig. 5 illustrates the ion concentration variation of SBF solution over

immersion periods. The obtained trend stated the process of ion ex­
change which had occurred between SBF solution and immersed BGs. A
similar trend in changes in concentrations of Si and Ca ions indicated an
upward trend until the third day and then a sudden change to a down­
ward one. According to the results of XRD, FTIR, and SEM, this reduction
belonged to the formation of the HA layer and decrease in concentration
of Si and Ca ions in the SBF solution. The formation of HA particles was
also observed in the intensified decreasing trend of P ion concentration.
Similar trends were observed on ion concentrations of the Zr-BGs in the
former research [46] and also silicate-based BGs with low Mg content
(1-5 mol.%) [57].

3.6. pH monitoring

Fig. 6 demonstrates the pH values of the SBF solution before and after
various soaking periods. It can be stated that by the increase of the Mg
amount, the pH value decreased and the lowest measured pH values at Fig. 5. The concentration of Si, Ca, P, Zr and Mg ions in SBF solution after
various immersion periods.
the end of the same immersion period were dedicated to ZBG-M7.
Furthermore, the relationship between the pH variety and the forma­
tion of HA was interpreted as follows that after the formation of HA, the

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A. Moghanian et al. Journal of Non-Crystalline Solids 554 (2021) 120613

Fig. 6. The pH variety of the SBF solution after various soaking periods.

increasing trend of pH values was changed to constant. This trend was

Table 4
the same as the one observed through the investigations on Zr-BGs [46].
The Vickers hardness of the as-synthesized BGs and after 7 and 14 days of
immersion.
HV (Vickers (kg/mm2)) 3.7. Mechanical properties
Specimens Pre-immersion After 7 days After 14 days

ZBG-M0 415±7 152±6 135±5

The measured HV (Vickers) values are reported in Table 4. As a
ZBG-M1 419±7 164±4 141±5 matter of fact, the the least and the highest Vicker hardness was for the
ZBG-M3 434±8 172±5 147±6 control specimen and the specimen with 7 (mol.%) Mg content. It was
ZBG-M5 441±10 178±6 156±8 visible that specimens with more Mg content revealed higher Vicker
ZBG-M7 453±11 183±7 162±7
hardness. It can be stated that, by increase of the immersion period (7
and 14 days) and as a result of more biodegradation, the HV values were

Fig. 7. The cell proliferation of osteoblast-like cells after co-culturing with synthesized BGs before, after 7 and 14 days of culture (*p< 0.05, **p< 0.01 and ***p
< 0.001).

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A. Moghanian et al. Journal of Non-Crystalline Solids 554 (2021) 120613

Fig. 8. The ALP activity of osteoblast-like cells after co-culturing with synthesized BGs before, after 7 and 14 days of culture (*p< 0.05, **p< 0.01 and ***p
< 0.001).

decreased. Moreover, in the previous paper, the positive effect of Zr on
network connectivity and as a result, increased compressive strength
[46]. Another reports, indicated the improved Vicker hardness of
silicate-based BGs under the influence of co-incorporation of Mg along
with other elements such as aluminium (Al) [58] and strontium (Sr)
[59].
3.8. In vitro biological evaluation
3.8.1. MTT assay
Fig. 7 demonstrates the MC3T3 cells proliferation based on the
measured OD through MTT assay. The following bar chart indicates the
positive effect of the addition of Mg on the function of the ZrBG with the
optimal amount of Zr (5mol.%) for stimulating cell proliferation and
differentiation. Moreover, statistical analysis of results revealed that

Fig. 9. The bactericidal percentage of synthesized BGs (*p< 0.05, **p< 0.01).

7
A. Moghanian et al.
Fig. 10. Dapi/Actin and Live/Dead fluorescence images of the ZBG-M3 and ZBG-M5 specimens after co-culturing with synthesized BGs for 7 and 14 days.
initially the performance of ZBG-M5 was significant toward ZBG-M0 and
ZBG-M3 (*p< 0.05, **p< 0.01). Comparing the values of ZBG-M0 and
ZBG-M1 on different days, it can be acknowledged that the addition of
1mol.% of Mg to the composition of Zr-BG did not have a statistically
significant effect (*p >0.05).On all days, the ZBG-M5 specimen was able
to perform better in comparison to other samples. On the other hand, the
ZBG-M7 sample showed a decrease in OD value in comparison to ZBG-
M5, under the influence of adding more Mg over 5 mol.%. However,
the statistical importance of its performance still indicated an
improvement in the function of the control Zr-BG (*p< 0.05).Although
the ZBG-M3 had lower performance than the ZBG-M5 sample, it was still
statistically significant as an upgraded ZBG (*p< 0.05). An incremental
trend of OD values of each specimen was also observed with increasing
the culturing period. Hence, the highest values were obtained on the
14th day of immersion. Some other reports were declared the positive
effect of Mg incorporation in 3.2 [60] and 3.5 [61] (wt.%) contents on
stimulating the proliferation of early osteoblast-like cells
3.8.2. ALP activity
The ALP activity of cells in the presence of synthesized BGs is
compared in Fig. 8. All specimens revealed more stimulating effect on
ALP activity of cell in comparison to the control sampl (*p < 0.05, **p <
0.01, and ***p < 0.001).The trend of ALP activity value was upward for
all specimens with an increase of culturing time. It can be stated that the
addition of 5 mol.% Mg content (ZBG-M5) significantly improved the
ALP activity of MC3T3 osteoblast-like cell line after 1, 7, and 14 days of
culturing. As demonstrated in Fig. 8, the highest value for ALP activity
was observed by the ZBG-M5, which contained 5 (mol.%) Zr and 5 (mol.
%) Mg in BG composition, after the 14th day of immersion.Similar to
MTT analysis, the results of ZBG-M3 were considered statistically sig­
nificant in comparison to the control specimen (**p < 0.01). Moreover,
it can be stated that the performance of the ZBG-M7 specimen was
weaker than the ZBG-M5 specimen due to the addition of Mg over 5 mol.
% (*p < 0.05). It was also reported that the addition of 8.5 and 8.9 (wt.
%) Mg was considered effectless while other reports stated the stimu­
lating effect for 3.5 (wt.%) [61] and 5 (mol.%) on ALP activity of cells
[57].
3.8.3. Antibacterial
In Fig. 9, the antibacterial percentage of the synthesized BGs is
shown. As the addition of Zr improved the antibacterial properties of
58S-BG in our previous work [46], the incorporation of Mg to the
structure of ZBG with the optimum amount of Zr (5 mol.%), was able to
contribute to the resistance of synthesized BG toward bacteria. As
8
Journal of Non-Crystalline Solids 554 (2021) 120613
perceived, ZBG-M3, ZBG-M5, and ZBG-M7 improved the bactericidal
performance of the control specimen which was considered statistically
significant (*p < 0.05, **p < 0.01). No similar significant differences
were observed of ZBG-M1 compared to sample one, in terms of statistical
analysis. The improved properties of silicate-based BG was also reported
by Zhang et al. [62] after the incorporation of 4.5 (wt.%) Mg content to
the composition of BG. The results of performed antibacterial studies on
Zr-BGs has also introduced the positive effect of Zr incorporation until 5
mol% [46]. Due to the superiority of the performance of ZBG-M3 and
ZBG-M5 in all bio-analysis, these samples can be introduced as modified
ZrBG with an optimal incorporation of Mg contents in composition.
3.8.4. Cell staining
Fig. 10 represents the biological performance of cells by utilizing
live/dead and dapi/actin cell staining after culturing along ZBG-M3 and
ZBG-M5.The increasing number of living cells versus the decreasing
number of dead cells indicated the excellent performance of ZBG-M3
and ZBG-M5.Also, better performance of ZBG-M5 in reducing dead
cells and increasing living cells can be seen compared to ZBG-M3. These
live/dead cell staining images confirmed and explained the results of
biological tests. Previously, Haimi et al. reported that the viable cells
number in Live/Dead assay was more in the specimens with 4–6 (wt.%)
Mg content in the composition of bioactive glass scaffolds in comparison
to zinc added specimens [63].
4. Conclusion
The study was about the design, synthesis, and characterization of
sol-gel derived silicate-based BGs in 60% SiO2-(31-X)% CaO-4% P2O5-
5% ZrO2 –X% Li2O (which X=0, 1, 3, 5 and 7 mol.%) system. The ability
of these BGs to form the HA layer through in vitro situation was
confirmed by the results of FTIR, SEM, and XRD analysis. In all speci­
mens, the HA particles were formed after the 7th day of immersion of
synthesized BGs in the SBF solution.This study also evaluated the
properties of synthesized BGs from the perspective of in vitro biology
along with mechanical to provide comprehensive research about these
BGs. On one hand, modified specimens revealed an improvement in ALP
activity and cell proliferation of MC3T3 osteoblast-like cells as well as
antibacterial function. This improvement was statistically evaluated and
proved the significant performance of ZBG-M3 and ZBG-M5 as the
specimens with 3 and 5 mol.% incorporation of Mg. Furthermore, co-
incorporation of the Mg along with Zr positively enhanced the me­
chanical properties of synthesized BGs, as the results of Vickers hardness
assay indicated.
A. Moghanian et al.
According to the results, ZBG-M3 and ZBG-M5 were introduced as
the reliable specimens for future in vivo experiments to reach multi­
functional BGs for tissue engineering and drug delivery.
Author contribution statements
• All authors discussed the results and contributed to the final
manuscript.
• A. Moghanian conceived and planned the idea of the research.
• A. Moghanian and M. Saghafi Yazdi supervised the project.
• M. Zohourfazeli and M. Tajer developed the theory and carried out
the experiment.
• M. Zohourfazeli, Z. Miri and M. Tajer wrote the manuscript with
support from A. moghanian.
• M. Zohourfazeli and M. Tajer contributed equally to this work.
• Z. Miri and M. Saghafi Yazdi done the complementary reviews.
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.
References
[1] A. Saatchi, A.R. Arani, A. Moghanian, M. Mozafari, Cerium-doped bioactive glass-
loaded chitosan/polyethylene oxide nanofiber with elevated antibacterial
properties as a potential wound dressingctive glass in tissue engineering, Ceram.
Int. (2020) https://doi.org/10.1016/j.ceramint.2020.12.078.
[2] T.H. Qazi, D.J. Mooney, M. Pumberger, S. Geissler, G.N. Duda, Biomaterials based
strategies for skeletal muscle tissue engineering: existing technologies and future
trends, Biomaterials 53 (2015) 502–521.
[3] A. Saatchi, A.R. Arani, A. Moghanian, M. Mozafari, Synthesis and characterization
of electrospun cerium-doped bioactive glass/chitosan/polyethylene oxide
composite scaffolds for tissue engineering applications, Ceram. Int. 47 (2020)
260–271.
[4] P. Kumar, B.S. Dehiya, A. Sindhu, Bioceramics for hard tissue engineering
applications: A review, Int. J. Appl. Eng. Res. 5 (2018) 2744–2752.
[5] L.L. Hench, Bioactive ceramics, Annals of the New York Acad. Sci. 523 (1988)
54–71.
[6] L.L. Hench, R.J. Splinter, W. Allen, T. Greenlee, Bonding mechanisms at the
interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 5 (1971) 117–141.
[7] M. Erol-Taygun, K. Zheng, A.R. Boccaccini, Nanoscale bioactive glasses in medical
applications, Int. J. Appl. Glass Sci. 4 (2013) 136–148.
[8] L.L. Hench, G. LaTorre, Reaction kinetics of bioactive ceramics Part IV: Effect of
glass and solution composition, Bioceramics 5 (1992) 67–74.
[9] L. Hench, A. Clark, H. Schaake, Effects of microstructure on the radiation stability
of amorphous semiconductors, J. Non-Crystal. Solids 8 (1972) 837–843.
[10] A. Moghanian, F. Sharifianjazi, P. Abachi, E. Sadeghi, H. Jafarikhorami, A. Sedghi,
Production and properties of Cu/TiO2 nano-composites, J. Alloys Comp. 698
(2017) 518–524.
[11] F. Shojaeepour, P. Abachi, K. Purazrang, A.H. Moghanian, Production and
properties of Cu/Cr2O3 nano-composites, Powder Tech. 222 (2012) 80–84.
[12] N. Salandari-Jolge, A.A. Ensafi, B. Rezaei, A novel three-dimensional network of
CuCr2O 4/CuO nanofibers for voltammetric determination of anticancer drug
methotrexate, Anal. Bioanal. Chem. (2020) 1–11.
[13] X. Zhang, Y. Yu, D. Jiang, Y. Jiao, Y. Wu, Z. Peng, J. Zhou, J. Wu, Z. Dong,
Synthesis and characterization of a bi-functional hydroxyapatite/Cu-doped TiO2
composite coating, Ceram. Int. 45 (2019) 6693–6701.
[14] A. Pazhouheshgar, A. Moghanian, S.A. Sadough Vanini, The extended finite
element method numerical and experimental analysis of mechanical behavior of
polysulfone/58s bioactive glass synthesized through solvent casting method,
Modares Mech. Eng. 20 (2020) 2061–2073.
[15] A. Moghanian, S. Firoozi, M. Tahriri, Synthesis and in vitro studies of sol-gel
derived lithium substituted 58S bioactive glass, Ceram. Int. 43 (2017)
12835–12843.
[16] M. Tahriri, M. Del Monico, A. Moghanian, M.T. Yaraki, R. Torres, A. Yadegari,
L. Tayebi, Graphene and its derivatives: Opportunities and challenges in dentistry,
Mater. Sci. Eng. C 102 (2019) 171–185.
[17] A. Moghanian, M. Zohourfazeli, Investigation the in vitro and bactericidal
properties of magnesium and copper containing bioactive glasses, J. Adv. Mater.
Tech. (JAMT) 9 (2020) 1–15, https://doi.org/10.30501/JAMT.2020.195763.1041.
[18] A. Moghanian, M. Zohourfazeli, Comparative study on in vitro, physico-chemical
and antibacterial properties of 58S and 68S bioactive glasses synthesized by sol-gel
method, Adv. Process. Mater. 13 (2020) 17–30, https://www.sid.ir/en/Journal/Vi
ewPaper.aspx?ID=716955.
[19] D. Khorsandi, A. Moghanian, R. Nazari, G. Arabzadeh, S. Borhani, Personalized
medicine: regulation of genes in human skin ageing, Allergy Therapy 7 (2016) 2.
9
Journal of Non-Crystalline Solids 554 (2021) 120613
[20] F. Baino, E. Fiume, M. Miola, E. Verné, Bioactive sol-gel glasses: processing,
properties, and applications, Int. J. Appl. Ceram. Tech. 15 (2018) 841–860.
[21] N.F. Ibrahim, H. Mohamad, S.N.F.M. Noor, Characterization on melt-derived
bioactive glass powder from SiO2-CaO-Na2O-P2O5 system, J. Non-Crystal. Solids
462 (2017) 23–31.
[22] A. Moghanian, M. Zohourfazeli, M.H. Mahdi Tajer, Z. Miri, S. Hosseini,
A. Rashvand, Preparation, characterization and in vitro biological response of
simultaneous co-substitution of Zr+4/Sr+2 58S bioactive glass powder, Ceram. Int.
(2020). https://doi.org/10.1016/j.ceramint.2020.11.139.
[23] A. Moghanian, S. Firoozi, M. Tahriri, A. Sedghi, A comparative study on the in vitro
formation of hydroxyapatite, cytotoxicity and antibacterial activity of 58S
bioactive glass substituted by Li and Sr, Mater. Sci. Eng. C 91 (2018) 349–360.
[24] A. Moghanian, S. Firoozi, M. Tahriri, Characterization, in vitro bioactivity and
biological studies of sol-gel synthesized SrO substituted 58S bioactive glass, Ceram.
Int. 43 (2017) 14880–14890.
[25] M. Aminitabar, M. Amirhosseinian, M.J. Elsa, Synthesis and in vitro
characterization of a gel-derived SiO2-CaO-P2O5-SrO-Li2O bioactive glass, Int. J.
Chem. Mol. Eng. 13 (2019) 296–307.
[26] M. Elsa, A. Moghanian, Comparative study of calcium content on in vitro biological
and antibacterial properties of silicon-based bioglass, Int. J. Chem. Mol. Eng. 13
(2019) 288–295.
[27] S. Kargozar, F. Baino, S. Banijamali, M. Mozafari, Synthesis and physico-chemical
characterization of fluoride (F)-and silver (Ag)-substituted sol-gel mesoporous
bioactive glasses, Biomed. Glass. 5 (2019) 185–192.
[28] A. Pazhouheshgar, S.A.S. Vanini, A. Moghanian, The experimental and numerical
study of fracture behavior of 58s bioactive glass/polysulfone composite using the
extended finite elements method, Mater. Res. Express 6 (2019) 1–22.
[29] A.A. Gorustovich, J.A. Roether, A.R. Boccaccini, Effect of bioactive glasses on
angiogenesis: a review of in vitro and in vivo evidences, Tissue Eng. Part B Rev. 16
(2010) 199–207.
[30] A. Khazaei, S. Saednia, J. Saien, M. Kazem-Rostami, M. Sadeghpour, M.
K. Borazjani, F. Abbasi, Grafting amino drugs to poly (styrene-alt-maleic
anhydride) as a potential method for drug release, J. Brazil. Chem. Soc. 24 (2013)
1109–1115.
[31] A. Moghanian, R. Portillo-Lara, E. Shirzaei Sani, H. Konisky, S.H. Bassir, N. Annabi,
Synthesis and characterization of osteoinductive visible light-activated adhesive
composites with antimicrobial properties, J. Tissue Eng. Regener. Med. 14 (2020)
66–81.
[32] M. Kazem-Rostami, A. Moghanian, Hünlich base derivatives as photo-responsive
Λ-shaped hinges, Org. Chem. Front. 4 (2017) 224–228.
[33] M. Diba, F. Tapia, A.R. Boccaccini, L.A. Strobel, Magnesium-containing bioactive
glasses for biomedical applications, Int. J. Appl. Glass Sci. 3 (2012) 221–253.
[34] Z. Huan, J. Zhou, J. Duszczyk, Magnesium-based composites with improved in
vitro surface biocompatibility, J. Mater. Sci. Mater. Med. 21 (2010) 3163–3169.
[35] S. Dutta, K.B. Devi, S. Gupta, B. Kundu, V.K. Balla, M.J. Roy, Mechanical and in
vitro degradation behavior of magnesium-bioactive glass composites prepared by
SPS for biomedical applications, J. Biomed. Mater. Res. Part B Appl. Biomater. 107
(2019) 352–365.
[36] S. Watts, R. Hill, M. O’donnell, R. Law, Influence of magnesia on the structure and
properties of bioactive glasses, J. Non-Crystal. Solids 356 (2010) 517–524.
[37] Z. Hajifathali, M. Amirhosseinian, The effect of substitution of CaO/MgO and CaO/
SrO on in vitro bioactivity of sol-gel derived bioactive glass, Int. J. Biomed. Biol.
Eng. 13 (2019) 279–287.
[38] J. Tamura, K. Kawanabe, M. Kobayashi, T. Nakamura, T. Kokubo, S. Yoshihara,
T. Shibuya, Mechanical and biological properties of two types of bioactive bone
cements containing MgO-CaO-SiO2-P2O5-CaF2 glass and glass—ceramic powder,
J. Biomed. Mater. Res. Offic. J. Soc. Biomater. Jpn. Soc. Biomater. 30 (1996)
85–94.
[39] A. Moghanian, A. Ghorbanoghli, M. Kazem-Rostami, A. Pazhouheshgar, E. Salari,
M. Saghafi Yazdi, T. Alimardani, H. Jahani, F. Sharifian Jazi, M. Tahriri, Novel
antibacterial Cu/Mg-substituted 58S-bioglass: Synthesis, characterization and
investigation of in vitro bioactivity, Int. J. Appl. Glass Sci. 11 (2019) 685–698.
[40] F. Sharifianjazi, M. Moradi, A. Abouchenari, A.H. Pakseresht, A. Esmaeilkhanian,
M. Shokouhimehr, M.S.J. Asl, Effects of Sr and Mg dopants on biological and
mechanical properties of SiO2–CaO–P2O5 bioactive glass, Ceram. Int. (2020).
[41] M. Prabhu, K. Kavitha, P. Manivasakan, V. Rajendran, P.J. Kulandaivelu, Synthesis,
characterization and biological response of magnesium-substituted nanobioactive
glass particles for biomedical applications, Ceram. Int. 39 (2013) 1683–1694.
[42] Z. Tabia, K. El Mabrouk, M. Bricha, K. Nouneh, Mesoporous bioactive glass
nanoparticles doped with magnesium: drug delivery and acellular in vitro
bioactivity, RSC Adv. 9 (2019) 12232–12246.
[43] H. Harianawala, M. Kheur, S. Kheur, T. Sethi, A. Bal, M. Burhanpurwala, F. Sayed,
Biocompatibility of zirconia, J. Adv. Med. Dent. Sci. Res. 4 (2016) 35.
[44] T. Ramesh, M. Gangaiah, P. Harish, U. Krishnakumar, B.J. Nandakishore, Zirconia
ceramics as a dental biomaterial–an over view, Trend Biomater. Artif. Organs
(2012) 26.
[45] M. Montazerian, B.E. Yekta, V.K. Marghussian, C.F. Bellani, R.L. Siqueira, E.
D. Zanotto, Bioactivity and cell proliferation in radiopaque gel-derived
CaO–P2O5–SiO2–ZrO2 glass and glass–ceramic powders, Mater. Sci. Eng. C 55
(2015) 436–447.
[46] A. Moghanian, M. Zohourfazeli, M.H.M. Tajer, The effect of zirconium content on
in vitro bioactivity, biological behavior and antibacterial activity of sol-gel derived
58S bioactive glass, J. Non-Crystal. Solids 546 (2020), 120262.
[47] M. Prabhu, K. Kavitha, S. Sutha, P. Manivasakan, V. Rajendran, P. Kulandaivelu, K.
J. Alameh, Bioactivity of zirconium-substituted nanobioactive glass particles,
Synthesis Reactivity in Inorganic, Metal-Org. Nano-Metal Chem. 45 (2015) 92–96.
A. Moghanian et al.
[48] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity?
Biomaterials 27 (2006) 2907–2915.
[49] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, Solutions able to
reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W3,
J. Biomed. Mater. Res. 24 (1990) 721–734.
[50] H. Zhu, C. Hu, F. Zhang, X. Feng, J. Li, T. Liu, J. Chen, J. Zhang, Preparation and
antibacterial property of silver-containing mesoporous 58S bioactive glass, Mater.
Sci. Eng. C 42 (2014) 22–30.
[51] H.M. Elgendy, M.E. Norman, A.R. Keaton, C.T. Laurencin, Osteoblast-like cell,
(MC3T3-E1) proliferation on bioerodible polymers: an approach towards the
development of a bone-bioerodible polymer composite material, Biomaterials 14
(1993) 263–269.
[52] M.C. Enright, D.A. Robinson, G. Randle, E.J. Feil, H. Grundmann, B.G. Spratt, The
evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA), Proc.
Natl. Acad. Sci. 99 (2002) 7687–7692.
[53] S. Hu, C. Ning, Y. Zhou, L. Chen, K. Lin, J. Chang, Antibacterial activity of silicate
bioceramics, J. Wuhan Univ. Tech.-Mater. Sci. Ed. 26 (2011) 226–230.
[54] S. Hu, J. Chang, M. Liu, C. Ning, Study on antibacterial effect of 45S5 Bioglass®,
J. Mater. Sci. Mater. Med. 20 (2009) 281–286.
[55] M. Zohourfazeli, M.H. Mahdi Tajer, A. Moghanian, Comprehensive investigation
on multifunctional properties of zirconium and silver co-substituted 58S bioactive
glass, Ceram. Int. (2020). https://doi.org/10.1016/j.ceramint.2020.09.093.
[56] M.d.M. Pereira, A. Clark, L. Hench, Calcium phosphate formation on sol-gel-
derived bioactive glasses in vitro, J. Biomed. Mater. Res. 28 (1994) 693–698.
10
Journal of Non-Crystalline Solids 554 (2021) 120613
[57] A. Moghanian, A. Sedghi, A. Ghorbanoghli, E. Salari, The effect of magnesium
content on in vitro bioactivity, biological behavior and antibacterial activity of
sol–gel derived 58S bioactive glass, Ceram. Int. 44 (2018) 9422–9432.
[58] B. Karakuzu-Ikizler, P. Terzioğlu, Y. Basaran-Elalmis, B.S. Tekerek, S. Yücel, Role of
magnesium and aluminum substitution on the structural properties and bioactivity
of bioglasses synthesized from biogenic silica, Bioact. Mater. 5 (2020) 66–73.
[59] D. Bellucci, A. Sola, R. Salvatori, A. Anesi, L. Chiarini, V. Cannillo, Role of
magnesium oxide and strontium oxide as modifiers in silicate-based bioactive
glasses: Effects on thermal behaviour, mechanical properties and in-vitro
bioactivity, Mater. Sci. Eng. C 72 (2017) 566–575.
[60] A. Saboori, M. Sheikhi, F. Moztarzadeh, M. Rabiee, S. Hesaraki, M. Tahriri,
N. Nezafati, Sol-gel preparation, characterisation and in vitro bioactivity of Mg
containing bioactive glass, Adv. Appl. Ceram. Struct. Funct. Bioceram. 108 (2009)
155.
[61] A. Saboori, M. Rabiee, F. Moztarzadeh, M. Sheikhi, M. Tahriri, M. Karimi,
Synthesis, characterization and in vitro bioactivity of sol-gel-derived
SiO2–CaO–P2O5–MgO bioglass, Mater. Sci. Eng. C 29 (2009) 335–340.
[62] D. Zhang, O. Leppäranta, E. Munukka, H. Ylänen, M.K. Viljanen, E. Eerola,
M. Hupa, L. Hupa, Antibacterial effects and dissolution behavior of six bioactive
glasses, J. Biomed. Mater. Res. Part A Offic. J. Soc. Biomater. 93 (2010) 475–483.
[63] S. Haimi, G. Gorianc, L. Moimas, B. Lindroos, H. Huhtala, S. Räty, H. Kuokkanen,
G.K. Sándor, C. Schmid, S. Miettinen, Characterization of zinc-releasing three-
dimensional bioactive glass scaffolds and their effect on human adipose stem cell
proliferation and osteogenic differentiation, Acta Biomater. 5 (2009) 3122–3131.