International Journal of Biological Macromolecules 137 (2019) 1256–1267

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International Journal of Biological Macromolecules

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

Alginate-bioactive glass containing Zn and Mg composite scaffolds for

bone tissue engineering
Delaram Zamani* , Fathollah Moztarzadeh, Davood Bizari
Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran

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

Article history: Past researches on bone regeneration field have shown the positive impacts of the presence of Zinc and
Received 1 January 2019 Magnesium ions in the bioactive glasses composition. However, there is no dedicated work on the effect
Received in revised form 16 June 2019 of the aforementioned bio-glass on the polymer matrix composites. The key idea of the approach is to
Accepted 24 June 2019 improve antibacterial efficacy, biological activity and mechanical properties of the bone composite scaffolds
Available online 4 July 2019
by incorporating bioactive glasses containing Zinc and Magnesium into alginate networks. The prepared
scaffolds were characterized by SEM, ATR-FTIR and XRD analysis. Compression strength of obtained highly
Keywords: porous composite scaffolds was remarkably enhanced by the presence of bio-glass particles. The maxi-
Bone tissue engineering
mum compressive strength (1.7 MPa) was obtained for alginate composite containing 1 g Mg-Zn-BG. In
Bioactive glass
vitro evaluation such as swelling, bio-mineralization, biodegradation were carried out, which indicates that
Alginate
incorporation of bio-glass promotes apatite deposition on composite scaffolds. Cytotoxicity, cell attachment
and proliferation and osteogenic differentiation were also evaluated by culturing MG-63 cells on scaffolds.
ICP analysis were conducted after 60 days of incubation in PBS solution to verify the ion release capability
of the composite scaffolds, particularly Zn and Mg ions, which resulted in significant antibacterial efficacy
enhancement of composite scaffolds against E. coli and S. aureus bacteria.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction from brown algae. Chemically, alginate belongs to a family of linear

Implantation and transplantation are the clinical methods for
repairing bone defects and injuries. However, due to the princi-
pal problems of transplantation and implantation such as lack of
donors in spite of a large number of applicants and the possibil-
ity of rejection or disease transmission, as well as the necessity
of revision surgery in implantation cases, tissue engineering con-
sidered as a new approach to repair and regenerate damaged tis-
sues [1]. Bone tissue engineering (BTE) has attracted attentions due
to bone’s self-regeneration ability under physiological conditions,
while in large bone defects it needs some structural or mechan-
ical support to heal completely [2]. The bone matrix consists of
organic component (collagen) reinforced by inorganic component
(hydroxyapatite). Various natural and synthetic materials or their
composites have been studied to develop this field [3]. In particu-
lar, biodegradable natural polymers due to their significant qualities
like biocompatibility, biodegradability, non-toxicity, as well as abun-
dance, have attracted attentions recently. For example, alginate is
a natural polysaccharide found in seaweeds and mainly extracted
* Corresponding author.
E-mail address: d.zamani@aut.ac.ir (D. Zamani).
polymeric acid composed of (1-4)-linked b-D-mannuronic acid (M
units) and (1-4)-linked a-l-guluronic acid (G units) residues. Among
natural polymers, alginate is widely used in the food and pharma-
ceutical industry as well as tissue engineering material. Alginate
shows good scaffold formation since its ability to form hydrogel
in the presence of certain divalent cations (e.g., Ca2+ , Sr2+ and
Ba2+ at low concentrations). On the other hand, alginate is highly
hydrophilic; as a result, it could be easily dissolved in aqueous solu-
tions so it shows weakness to support structures. To overcome this
problem, alginate scaffolds are crosslinked by divalent-cations-based
substances notably calcium-based substances like calcium chloride
(CaCl2 ) [4–6].
In spite of the reports about the use of polymers in bone tissue
engineering, unfortunately, they show shortcomings and disadvan-
tages. For instance, polymers’ mechanical properties and flexibility
are not appropriate as bone scaffolds [7]. Another limitation is that
it does not induce bio-mineralization, which is necessary for bone
regeneration. In order to overcome these weak points, new bioma-
terials are designed which significantly improve interactions with
surrounding tissues. In order to contribute strength to ductile organic
components, the effect of incorporation of mineral components has
been investigated in the literature. Some of the well-known mineral
components, which have been focused on, are bio-ceramics and

https://doi.org/10.1016/j.ijbiomac.2019.06.182
0141-8130/© 2019 Elsevier B.V. All rights reserved.
 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267
bioactive glasses’ fillers or coatings. These two features, namely duc-
tility and strength, lead to high energy absorption before failure [8,9].
In bone regeneration, bioactive glasses are more impressive than
bio-ceramics because of their significant advantages which can be
regarded as the ability to form a strong bond to both hard and soft
tissues and also gene expression in osteoblast cells and angiogenesis
by the bio-glasses’ dissolution products. Although bio-glass scaffolds
are widely investigated due to their osteoconductive properties, they
should preferably used as a second phase of the polymer compos-
ites or as implant’s coating because of their fast dissolution behavior
and fragility [10–13]. Hench and coworkers made the first bioactive
glasses in the 1970s which consisted of 4 components (46.1 mol.%
SiO2 , 24.4 mol % Na2 O, 26.9 mol % CaO and 2.6 mol %P2 O5 ) which is
currently known as 45S5 and Bioglass® [14]. Bioactive glasses can
be prepared by sol-gel synthetic technique. One positive aspect of
using this method is the ability to control the structure, surface area
and glass composition [8,15]. Bioactivity of these glasses depends
on their composition, for this reason the effect of various additives
which often are chosen among human body trace elements like Zinc
(Zn), Magnesium (Mg), Aluminum (Al), Born (B), Strontium (Sr) as
well as Cobalt (Co) have been studied [16]. Researches demonstrate
that such trace elements improve the interactions between bone and
cells. Among these essential trace elements, both Zink and Magne-
sium play a vital role in bone metabolism by enhancing alkaline
phosphatase activity [17].
Although bioglasses have been used as bone graft expander,
some surgeons declared that the lack of antibacterial efficacy is
still their drawback and being cited as a significant area of con-
cern [11]. It would be beneficial if a synthetic composite bone graft
was built based on elements with antibacterial nature which can
minimize the risk of primary infection. Zinc ions show antimicro-
bial and anti-inflammatory properties as well as having stimulatory
effect on bone formation. Consequently, regeneration takes place
under bacteria−free conditions [18]. Researches have also shown
that bone tissue contains about 0.0126 –0.0217 wt. % Zn element and
the deficiency of Zinc may cause osteoporosis in old people [11].
More impressive outcomes observed by doping Mg elements with
inherent antibacterial properties into glass composition. Mg ion
which is the fourth most abundant positively charged ion in the
human body, stimulates Stem Cells proliferation and differentia-
tion. Moreover, Magnesium plays a vital role in providing the cell
apoptosis control. It also improves the mechanical properties of
newly formed bone [16,19,20]. In addition, the mechanical proper-
ties of bio-glass improved by adding zinc and magnesium elements
to bio-glass composition which can be attributed to the higher bond
energy of Mg-O and Zn-O compared to Ca-O [10,11]. Consequently,
bone’s formation of patient could be accelerated by incorporation
of suitable amount of Mg and Zn into the hard tissue implants’
composition.
Apart from positive effects of doping Zn and Mg ion into glass
composition, it has reported in the literature that it may cause
disadvantages like retarding the nucleation rate of apatite forma-
tion and inhibiting the transformation of amorphous apatite into
crystalline carbonated hydroxyapatite [19,21–23]. A possible expla-
nation is substitution of CaO with ZnO and MgO in the bioactive
glass, while lower calcium content can result in reduced apatite for-
mation [24]. With the above consideration, in our study we use
alginate crosslinked by CaCl2 as polymer matrix. By this means, the
lack of Ca ions will be compensated during alginate degradation.
Several papers have studied about the incorporation of hydroxyap-
atite, bioglass, calcium phosphate and silica as a second phase into
alginate polymer matrices [4,12,13,25–28]. Despite of the proven
positive results of doping Zinc and Magnesium additives, to the best
of author’s knowledge, no paper in the literature has heretofore
dealt with the effect of incorporating of these novel bio-glasses into
polymers.
1257
The main contribution of this paper is to evaluate the influence of
Mg and Zn containing bioactive glass on alginate hydrogel and exam-
ine physical, mechanical, and biological properties of this composite
scaffold for bone tissue regeneration. The key feature of this class of
scaffolds is releasing essential trace elements, i.e., Zn and Mg, which
have been proved to have antibacterial effect and improve mechan-
ical properties of scaffold. Despite other studies in the literature in
which the presence of Mg and Zn in bioglass composition at the
expense of Ca results in reduced apatite formation, in this paper,it is
expected to observe good biomineralization due to releasing Ca ions
during alginate degradation.
The remainder of this paper is organized as follows. In the first
section, materials and methods used to synthesis Mg-Zn-BG as well
as fabricate composite scaffolds are fully addressed. In the subse-
quent section, the effect of Mg-Zn-BG content on the physicochem-
ical, mechanical and biological properties of composite scaffolds are
discussed. Finally, the paper concludes with some remarks and hints
as ongoing works.
2. Materials and methods
In this section, the synthesis of bioactive glass and fabrication of
composite scaffolds of Alg/BG will be discussed as follows:
2.1. Synthesis of bioactive glass ceramic particles
The synthesis procedure of bioactive glass ceramic containing
zinc and magnesium by sol-gel method was reported in our previous
studies [12]. The synthesis of the glass with composition of SiO2 -
P2 O5 -CaO-ZnO-MgO (60%SiO2 , 26%CaO, 4%P2 O5 , 5%ZnO, 5%MgO, mol
%) was performed as follows: 9.7 ml of 2N HNO3 which is an acid
hydrolysis catalyst, was added to 58.2 ml of stirring distilled water
at room temperature. Thereafter, 53 ml of tetraethyl orthosilicate
(TEOS) was added to the solution, stirred up to 60 min (molar ratio
of the sum of TEOS and TEP to H2 O was 6 to 1 and molar ratio of
HNO3 to H2 o was 12 to 1). The end of hydrolysis can be detected
by achieving a transparent liquid inside the container. Then, 24.601
gr of calcium nitrate tetrahydrate (Ca(NO3 )2 .4H2 O), 5.4 ml of tri-
ethyl phosphate (TEP, 99.8%), 5.137 g of magnesium nitrate hexahy-
drate (Mg(NO3 )2 • 6H2 O) and 5.96037 g of Zinc nitrate hexahydrate
(Zn(NO3 )2 .6H2 O was added separately while allowing 30 minutes
for each reagent to completely dissolve. The mixture aged for three
days at 37◦ C to obtain a white gel. The gels were crushed by spatula,
to make the next step (drying) more efficient. Differential Thermal
Analysis and Thermo Gravimetric analyses (DTA/TG) were used to
determine the appropriate time and temperature for this process.
Thermogravimetric analysis of the synthesized bio-glass was moni-
tored by using TG/DTA instrument with a heating rate of 5 ◦ C/min
and a temperature range of 25–1000◦ C. The results of the latter anal-
ysis is reported in [12]. Subsequently, the gels were dried at 120◦ C
for 24 h followed by ball milling for 10 min at a speed of 200 rpm.
The obtained powders were stabilized at 650◦ C for 3 h. The extracted
powders were re-grinded to reach the suitable size, in the range of
15–60 nm.
2.2. Fabrication of Alg/BG composite scaffolds
Sodium alginate (Sigma Aldrich, Germany), calcium chloride
(Merck, USA), bioactive glass (BG) particles containing Zinc and Mag-
nesium (15–60 nm particle size) and deionized water were used
to prepare the composite scaffolds by freeze-drying method. To
achieve this goal, pure alginate scaffold as well as the bio-composite
scaffolds containing bioactive glass and alginate with different pro-
portions were prepared. The weight composition of bioactive glass
and alginate in the samples is reported in Table 1. Briefly, to pre-
pare pure alginate scaffold, 3% sodium alginate gel was prepared
1258 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267

Table 1
Labels used for different samples as a function of their composition.
Sample Alginate powder (g) Bio-glass powder (g)
Pure alginate scaffold 3 0
Alg-0.3BG 3 0.3
Alg-1BG 3 1
Alg-1.5BG 3 1.5
and in order to fabricate composite scaffolds, the required weighed
quantity of bioactive glass powders regarding as reported in Table 1
dispersed in 100 ml distilled water by magnetic stirring. Meanwhile,
the sodium alginate powder was added to the solution and was kept
stirring for 6 h at room temperature. The resulting gel was poured
into 10 cm diameter plates and 1 cm diameter cylindrical molds and
they were stored in a freezer at −20◦ C overnight. Subsequently,
they were lyophilized by freeze dryer at −80◦ C for 2 days. The dried
samples were crosslinked by immersing in 5%(w/v) CaCl2 solution
for 20 min followed by washing and immersing in distilled water.
Finally, the scaffolds were refreeze-dried for 6 h to obtain dried
composite scaffolds. Fig. 1 demonstrates the process of fabricating
composite scaffolds.
2100). Specimens were cut by a sharp blade and were coated with a
thin layer of gold. Image j software was used to determine pore size.
The average pore size was determined by averaging the sizes of 25
voids.
2.3.4. Porosity and density measurement
The porosity and density of scaffolds were measured according to
a liquid displacement method. The dry scaffolds of 2 × 2 cm2 was
weighted (Wi ) and placed in a determined volume of ethanol (V1 ) for
15 min for voids to be filled completely by ethanol. The new volume
which is the summation of ethanol and the scaffold itself is denoted
by V2 . Then scaffolds were weighted again (Wf ) while the remain-
ing volume of ethanol was noted V3 . Ethanol with the density of
0.789 was used due to its ability to fill the pores effortlessly while it
is not absorbed by the scaffold. Five specimens were examined for
each sample of polymer and composite scaffolds and the results were
averaged. Porosity and density were calculated according to Eqs. (1)
and (2):
(Wf −Wi )
qethanol
%Porosity = × 100 (1)
V2 − V3

2.3. Physico-chemical and structural characterizations

Wi
%Density = (2)
V2 − V1
In this section, the inter-molecular interactions, structural mor-
phology, porosity, and density of scaffolds will be discussed.
2.4. In-vitro swelling studies
2.3.1. Attenuated Total Reflection Fourier Transform Infra-Red
(ATR-FTIR) spectroscopy The water uptake ability of scaffolds with defined weight (Wd )
Inter-molecular interactions of scaffolds’ components in the com- was measured by soaking them in Phosphate Buffered Solution (PBS)
posite scaffolds, alginate, and BG were characterized by ATR-FTIR for 1, 3, 6, 12 and 24 h. The hydrogels were removed and weighted
spectrometer (Thermo Scientific Co.). The spectra were recorded wet (Ww ) after removing the excess water by surface wiping. Four
ranging from 650 to 4000 cm−1 . specimens of each composition sample were measured and the
average value was considered as the water uptake ability. Water
2.3.2. X-Ray Diffraction (XRD) analysis absorption (WA ) was calculated according to the following equation:
The XRD patterns of Alg/BG scaffolds were obtained by analyti-
cal XPERT PRO powder diffractometer (Cu K radiation) operating at Wi − W d
WA = (3)
room temperature and 40 kV and 30 mA voltage and current division. Wd
XRD was taken at 2h angle range of 3–118.
2.5. Mechanical testing
2.3.3. Scanning Electron Microscopy (SEM)
The structural morphology and pore size of scaffolds were exam- As the compression test is the most relevant test for replace-
ined by scanning electron microscope (SEM-Seron Technology-AIS ment of cancellous bone [29], the compression test were performed

Bioactive
Glass Powder
Alginate containing
3% Wt Mg & Zn

Lyophilized

Cross-linked
with CaCl2

Fig. 1. Schematic diagram describing the fabrication of Alginate-bioactive glass composite scaffold.
 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267
on cylindrical samples of 1 and 1.5 cm in diameter and height. Uni-
versal mechanical tester (SANTAM-STM 1) was used in a z-direction
with crosshead speed of 1 mm/min at room temperature. Each sam-
ple was compressed to approximately 20% of its initial height. The
mechanical properties consisting of toughness, elastic modulus and
compressive strength at two different elongations of 30% and 70%
were calculated. While toughness was measured by integrating the
stress-starin curves, elastic module was determined by the slope of
the initial linear region. Testing was carried out in triplicate and the
results were illustrated as mean value ± standard deviation.
2.6. In-vitro degradation studies
A degradation study of the scaffolds was carried out in PBS solu-
tion at pH 7.4 in 37 ◦ C for 3, 7, 14, 21, and 30 and 60 days. Initially,
the weight of each specimen was measured accurately (Wi ). There-
after, samples were withdrawn from the media at prescribed times
and then washed carefully with deionized water to remove ions
absorbed on the surface and freeze-dried. Two third of the media
was refreshed every three days. Final weight of the dried samples
was denoted by Wf . The degradation ratio was calculated using the
following equation:
W i − Wt
Degradation Ratio= × 100
Wt
Three specimens were analyzed for each scaffold composite sample
and the average value is reported.
In order to evaluate the concentration of different released ions
of alginate matrix and bio-glass particles such as Ca, Si, P and in
particular, Zn and Mg, the alginate and composite scaffolds were
soaked in PBS for 60 days and the resulting solutions were analyzed
using inductively coupled plasma-optical emission spectroscopy
(ICP-OES). In order to exclude concentration of aforementioned ions
in fresh PBS from obtained results, fresh PBS was set as a control
solution in the test.
2.7. In-vitro antibacterial evaluation
The antimicrobial activity of pure alginate and composite scaf-
folds were evaluated against Gram positive S. aureus (ATCC®
25923TM ) and Gram negative E. coli (ATCC® 15597TM ) using a liq-
uid culture method. Bacteria were cultured in liquid lysogenybroth
(LB) medium overnight at 37◦ in an orbital shaker. Before the exper-
iment, the bacterial suspension were diluted to approximately 108
CFU/mL and the alginate and composite scaffolds (each with 10 mg
weight) were sterilized by UV lamp for one hour at room tempera-
ture. Then, the scaffolds were incubated overnight at 37◦ and shaken
at 100 rpm with 1 ml of the bacterial suspension. The optical den-
sities (OD) of the bacterial solution including one of the scaffolds
(ODs ) were measured after 12 h at 600 nm and the results compared
with the OD of the bacterial LB broth solution (ODb ) as control. The
bacterial inhibition could be calculated by the following equation.
ODb − ODs
Bacterial inhibition = × 100
ODb
2.8. In-vitro bio-mineralization behavior
The most reliable method to determine the bioactivity is in-vivo
studies but due to the animal welfare laws, such investigations are
avoided at an early stage of the study. Hence, the bioactivity of the
scaffolds was determined by immersing the samples in Simulated
Body Fluid (SBF) solution, which was prepared by using Kokubo’s
protocol [30]. The samples were immersed in 30 ml of SBF for pre-
scribed times of 3, 7, 14, and 21 days at 37◦ C. Afterwards, each
1259
sample rinsed in distilled water, freezed and lyophilized. Forma-
tion of hydroxyl apatite on the surface was examined through XRD
analysis and FESEM.
2.9. Cell culture
The MG-63 cell line obtained from Royan Institute of Iran
(CRL-1427, 14 years, Caucasian, Passage 4) was cultured in the
media consisting of Dulbecco’s Modified Eagle’s Medium (DMEM,
Bioidea, Iran), supplemented with 10%(v/v) fetal bovine serum (FBS,
Bioidea, Iran), 1% (v/v) sodium pyruvate solution and 1%(v/v) peni-
cillin/streptomycin (pen/strep, Bioidea, Iran). The scaffolds were
kept in an incubator at 37◦ C in a humidified atmosphere contain-
ing 5%CO2 , while the culture medium was changed routinely every
2 days. Before cell seeding, scaffolds were washed with phosphate
buffer saline (PBS, Bioidea, Iran). Sterilization of scaffolds was per-
formed by immersion in 70% ethanol for 30 min followed by UV light
for 2 h. After reaching 70% confluency, the cells were detached with
0.25% trypsin and were counted by trypan blue assay. Finally, a sus-
pension containing 30000 cells were seeded on the upper surface of
the scaffolds, which were soaked in media for 24 h in advance. Tis-
sue culture plate (TCP) was considered as control material. The cells
were seeded and cultured on scaffold’s surface and were left in the
(4) incubator at 37◦ C under 5% CO2 condition for 7 days and medium was
changed every three days.
2.10. Cell viability study
The cell viability was examined using MG-63 cells by MTT pur-
chased from Sigma-Aldrich colorimetric assay after 12, 24, and 72 h
of incubation. The culture medium was extracted from each well and
replaced by 500 ll of MTT solution (0.5 g /ml) and were incubated
at 37◦ C under 5% CO2 , for 6 h in order to form formazan prod-
ucts. Thereafter, Dimethyl Sulfoxide (DMSO) was added to dissolve
the dark blue formazan crystals. The DMSO absorbance was read at
wavelength of 490 nm by ELISA reader (Bio-Rad, model, USA), which
is proportional to the number of attached cells. Experiments were
carried out in triplicate per sample. All results were illustrated as the
mean ± standard deviation for n=3
2.11. Cell attachment and morphology study by SEM observation
The attachment and morphology of the cells seeded on the algi-
nate and composite scaffolds were evaluated by SEM analysis (SEM-
Seron Technology-AIS 2100). After 12, 24 and 72 h of culture, the
cells were fixed with 2.5% glutaraldehyde (Sigma) for three hours.
Glutaraldehyde solution extraction and dehydration of attached cells
were done by immersing in gradient ethanol solution of 30, 70,
90, 96, and 100%. At last, the cells attached to the scaffolds were
evaluated by SEM images.
2.12. Alkalin phosphatase activity
(5)
The osteogenic differentiation of the cells in the alginate and com-
posite scaffolds was measured by an alkalin phosphatase (ALP) assay.
The ALP activity of the cells were determined based on the hydroly-
sis of p-nitrophenyl phosphate to p-nitrophenol. The scaffolds were
washed with PBS, and MG-63 cells were seeded on the scaffolds and
incubated under standard condition using supplemented complete
medium for 7 and 14 days. Then, the scaffolds were washed with PBS
and lysed with 0.1% triton X-100 solution followed by sonication for
30 min. In the presence of ALP, p-nitrophenylphosphate was added
and the absorbance of ALP was measured at 405 nm.
1260 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267

40
38
36
34
32
30
28
26

Frequency (%)
24
22
20
18
16
14
12
10
8
6
4
2
0
0 50 100 150 200 250 300 350 400 450

Pore size ( m)

(a) (b)
36

32

28

Frequency (%)
24

20

16

12

0
0 50 100 150 200 250 300 350 400 450

Pore size ( m)

(c) (d)
32

28

24
Frequency (%)

20

16

12

0
0 50 100 150 200 250 300 350 400 450
Pore size ( m)

(e) (f)
32

28

24
Frequency (%)

20

16

12

0
0 50 100 150 200 250 300 350 400 450

Pore size ( m)

(g) (h)
Fig. 2. Cross-sectional SEM micrographs with markers which indicate well distribution of bioglass particles and corresponding pore size distribution histograms which reveal
two ranges of pore sizes for each of the samples, respectively 75 and 250 lm: (a) and (b) alginate scaffld, (c) and (d) scaffold sample Alg-0.3BG, (e) and (f) scaffold sample Alg-1BG
and (g) and (h) scaffold sample Alg-1.5BG.
 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267 1261

2.13. Statistical analysis
The data in this study were analyzed using one-way analysis
of variance (ANOVA) and reported as mean ± standard deviation.
Statistical significance was set at P-value < 0.05.
3. Result and discussion
3.1. Micro-structure of scaffolds
The morphology and micro-structure of the scaffolds were inves-
tigated due to the importance of porosity in bone tissue engineering.
Interconnected pores are essential to facilitate the nutrient flow and
create a framework for bone growth [31]. In [32], it has been claimed
that minimal acceptable pore size for bone tissue engineering is
almost 75 lm. Fig. 2 and show the cross section micrographs of the
pure alginate and composite scaffolds as well as the histograms of
the pore sizes which are estimated by image j software. Microstruc-
ture of scaffolds depends on different content of second phase and
the freeze drying’s factors like rate of freezing and temperature.
However, in this paper, the latter two factors were kept unchanged
during the process. Two ranges of pore size were observed for both
alginate and composite scaffolds, approximately 75 and 275 lm. It
has been reported that distribution of pore size could improve bone
cells function and is favorable for bone regeneration. While ultra-fine
pores enhance cell attachment and protein adhesion since most of
the cells are observed on nanometer scales, e.g., osteoblast cells (10–
30 lm average size), presence of larger pores at micrometer scales
Natural bone porosity is almost 70%. However, since some parts
of the scaffold are going to be filled with new tissues when implanted
in the body, the scaffolds are designed to reach over 80% porosity.
Fig. 4a demonstrates the porosity in the scaffolds. It can be seen that
scaffold’s porosity decreased slightly by increasing bio-glass content.
The density of the scaffolds determined by liquid (ethanol) displace-
ment method, as depicted in Fig. 4b, was ranged from 0.4 to 0.5 g /cm
3
. One of the main challenges in the scaffold fabrication is to make a
balance between its porosity and mechanical strength. The scaffold
strength will be discussed in detail in the following sections.
Three-dimension network scaffolds facilates the absorption of
nutrients for cell growth and metabolism. As a result, the scaffolds
should have the ability to absorb water and swell, instead of being
collapsed. It is proven that early swelling facilitates cell attachment
and proliferation [36,38]. Most natural polymers such as alginate
swell in biological fluids. In this study, the scaffolds swelled rapidly
within an hour and then the swelling rate dropped and reached an
almost constant value after 6 hours. As shown in Fig. 4c, after 24
hours, the swelling ratio of pure alginate scaffold is about 23%, which
is higher in comparison with the composite scaffolds. Water absorp-
tion ratio was decreased by adding bio-glass, which may attribute
to porosity and pore size decrease. In addition, increasing bio-glass
content will decrease polymer surface of the pore walls, which limits
the polymer chains ability to bind water molecules. Nevertheless, it
(e) Si-O-Si
(d)
Transmitance (a.u.)

is essential for cell migration and vascularization [33,34]. Referring (c)

to Fig. 2, the average pore size was decreased slightly by increasing
the bio-glass amount. A possible explanation is that the size of ice (b)

crystals formed in the freezing process maybe affected by the exis-

tence of bioglass due to the interaction bioglass crystals with water
molecules [35]. It was observed from Fig. 2 that alginate scaffold’s COO-(asym.) C-O-C
(a) OH COO-(sym.) C-C
pore walls are considerably smoother than the composite ones and
the scaffold pore shapes turn from circular to oval by increasing the
glass content. Some of the glass particles are indicated in Fig. 2. 4000 3500 3000 2500 2000 1500 1000
Wavenumber (cm-1)
3.2. Physicochemical properties
(a) ATR-FTIR spectra of a) alginate scaffold, b) composite scaf-
Nature of interaction between the alginate and bio-glass was
fold sample Alg-0.3BG, c) composite scaffold sample Alg-1BG, d)
examined by ATR-FTIR and XRD techniques. The ATR-FTIR spectra
obtained from bio-glass powder, pure alginate and alginate/bio- composite scaffold sample Alg-1.5BG and e) synthesized bioglass
glass composite scaffold are presented in Fig. 3a. The ATR-FTIR
particles
spectrum of pure alginate displays characteristic bands of peaks
around 1591 cm−1 and 1418 cm−1 corresponding to antisymmetric
and symmetric COO− stretching vibration of carboxylate salt groups
of alginate. It also shows adsorption bands at 3247 cm−1 , 1079 cm−1
and 1026 cm−1 , which correspond respectively to OH, C O C
intensity (a.u.)

and C C stretching bonds. The revealed results are consistent with (c)
those reported in the literature [31,36]. ATR-FTIR spectra of syn-
thesized bio-glass containing Zn and Mg showed a broad vibration
(b)
band located in the range of 900–1100 cm−1 which is assigned to
Si O Si asymmetric stretching band [24]. Comparing the ATR-
FTIR spectrum of composite Alg/BG with pure alginate scaffold and
pure bio-glass powders, suggests that characteristic bands of both (a)
alginate and bio-glass are present in composite scaffolds that con-
firms the interactions between alginate and bio-glass. Fig. 3b shows 20 40 60 80 100
XRD spectra for pure alginate, Alg/BG composite scaffold sample Alg- 2
1.5BG and bio-glass particles. Spectra of both alginate and bio-glass
showed wide peaks between 10 and 50◦ (2h) and between 20 and (b) The XRD patterns of a) pure alginate scaffold, b) composite
40◦ ; (2h), respectively, which demonstrates that they are both amor- scaffold sample Alg-1.5BG and c) synthesized bioglass particles
phous in nature. Only low intensity peaks at 17◦ in pure alginate (also
detected in previous studies) and at 31◦ in bio-glass spectra were Fig. 3. a) ATR-FTIR and b) XRD patterns of bioglass particles, alginate and composite
observed which does not imply crystallinity [37]. scaffolds.
1262 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267

should be noted that swelling behavior and porosity could affect the 3.0
Alginate scaffold
mechanical properties of the scaffolds. Thus, an appropriate water Alg-0.3BG
uptake is needed for bone tissue engineering application. 2.5 Alg-1BG
Alg-1.5BG

3.3. Mechanical properties 2.0

(MPa)
Research around bone tissue engineering has focused on how to 1.5
improve scaffolds strength in order to provide suitable structural
support during tissues regeneration. Compressive stress-strain curve 1.0
of the different composite scaffolds generally has three similar char-
acteristic stages. These stages can be regarded as an initial stage
0.5
attributed to the linear elasticity region at low strains (Hookean) due
to compression of cell walls, followed by a long plateau region in
0.0
which stresses do not vary significantly which represents plastic col- 0.0 0.2 0.4 0.6 0.8 1.0
lapse. Finally, due to almost complete cell collapse, the curves end
(%)
with a densification region in which the stress increased sharply with
strain [39,40]. However, most of highly porous polymer-based scaf- (a) The compressive stress strain curve of alginate and composite
folds manifest a continuous load increase instead of plateau region scaffolds
in the second stage because of constant porosity reduction and pore 1.8
20 1.2
30%
walls’ fracture resistance [41–43]. Mechanical properties of both Elastic modulus

Compressive strength (MPa)

1.6 18 1.1
70% Toughness
1.0
cylindrical pure alginate and composite scaffolds were evaluated 1.4 16
0.9

Elastic modulus (MPa)

Toughness (MJ/m3)
14
by plotting the engineering strain-stress curve which is depicted in 1.2
12
0.8
0.7
1.0
Fig. 5a and measuring the important properties like elastic module, 10 0.6
0.8 0.5
compression strength at two different elongations (s 30% and s 70% ) 0.6
8
0.4
6
and scaffold’s toughness as it can be seen in Fig. 5b and c. 0.4 4
0.3
0.2
Incorporating of the bio-glass should affect the reinforcement of 0.2
2 0.1
0 0.0
the scaffolds as stated by composite theories [44]. Furthermore, in Alginate Alg-0.3BG Alg-1BG Alg-1.5BG Alginate Alg-0.3BG Alg-1BG Alg-1.5BG
Samples
[10,11], it has been shown that due to higher band energy of Mg-O Samples

and Zn-O with respect to Ca-O, the mechanical properties of bio-glass (b) Mean values of the scaffolds’ compres- (c) Mean values of the scaffolds’ tough-
sive strength at two different deformation ness and elastic modulus obtained from
percentages compressive stress strain curve
100 0.6

Fig. 5. Mechanical properties plots of the scaffolds samples.

0.5
80
Density (gr/cm3)

0.4
Porosity (%)

60

0.3

40
0.2
will be improved in presence of Mg and Zn. Hence, it was expected
20
0.1 to observe an increasing trend of compressive strength by adding up
0 0.0
to 1.5 g of the glass containing Zn and Mg. Nevertheless, increasing
Alginate Alg-0.3BG Alg-1BG ALG-1.5BG Alginate Alg-0.3BG Alg-1BG ALG-1.5BG
the bioactive glass content could affect the formation and stabiliza-
Samples Samples
tion of polymer bands. In addition, the possibility of the bio-glass
(a) The porosity of samples consisting (b) The density of samples consisting particles agglomeration and inhomogeneous dispersion might prop-
various amounts of bioglass particles various amounts of bioglass particles agate polymer/bio-glass interfaces defects. In this paper, according to
the stress strain curves of the scaffolds compressed up to 80% which
24 is depicted in Fig. 5, scaffold sample Alg-1BG revealed the superior
mechanical properties among the others. The incorporation of bio-
22
glass up to 1 g(sample Alg-1BG) increased the compression strength
at 30% and 70% elongation (s 30% and s 70% ) from about 0.25 to 0.68
Swelling ratio (%)

20
and from 0.54 to 1.7 MPa, respectively. The values are similar to those
18
of cancellous bone(5–10 MPa [29]). However, by further increasing
of the bioglass content, aforementioned strength decreased to 0.39
16 and 0.87 MPa, respectively. Fig. 5c shows that the Young’s mod-
Alginate ulus of the scaffolds was noticeably improved by adding bioglass
14 Alg-0.3BG content. In other words, adding up to 1.5 g bio-glass powders to
Alg-1BG alginate scaffold resulted in more than fivefold increase in Young’s
12 Alg-1.5BG module from 3.5 to 18 MPa. In addition, as seen in Fig. 5a com-
posite scaffolds toughness improved from 0.24 MJ/m3 (pure alginate
10 scaffold) to 0.34 MJ/m3 (scaffold sample Alg-0.3BG) and 0.78 MJ/m3
0 5 10 15 20 25
(scaffold sample Alg-1BG). However, further increasing in bioglass
Time (hour)
content, led to toughness reduction to 0.45 MJ/m3 (scaffold sample
(c) Swelling studies of samples consisting various amounts of bio- Alg-1.5BG).
glass particles in PBS solution Although it is widely accepted that proper mechanical proper-
ties are required for bone tissue scaffolds, as they are mostly used
Fig. 4. a) Porosity, b) density and c) swelling ratio of alginate and alginate-bioglass for low load bearing applications such as oral and maxillofacial surg-
composite scaffolds. eries, bone defect filler, it is not essential to have high mechanical
 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267 1263

properties [41,45]. Considering the compressive strength of cancel-

lous bone (5–10 MPa) [29], as well as the the compressive strength of
highly porous biodegradable composites which were widely studied
for replacement of cancellous bone (∼0.015− ∼ 1 MPa) [46], it could
be concluded that the Alg/BG composite scaffold is appropriate to be
used to fill defects in cancellous bone.

3.4. Bio-mineralization

According to Kokubo’s approach, bioactivity of the composite

scaffolds was confirmed by formation of hydroxylapatite on the
surface after immersing samples in SBF solution at 37◦ C for 3, 7,
14 and 21 days [30]. Bioactive glasses manifest unique properties
by responding to the physiological fluids and forming a carbon-
ated hydroxyapatite layer (HCA). The main role of HCA layer is to (a) Alginate, 3 days (b) Alginate, 21 days
make a strong bonding between bioactive glasses and collagen fila-
ments produced by osteoblasts. The sequence of chemical reactions
which occurs to form this layer on the implant’s surface includes: (i)
exchange of calcium ions from surface with protons; (ii) silanol for-
mation produced on surface; (iii) polymerization of silanol groups
to produce amorphous silica gel; (iv) migration of calcium ions
and phosphate ions to surface to form apatite, (v) crystallization
of apatite. Lower Ca content in bioactive glass containing Zn and
Mg will result in decreased catione-proton exchange which leads
to lower apatite formation [24,47]. On the other hand, crosslinked
alginate degradation will release Ca ions which may compensate
the lack of Ca ions. The formation of hydroxyapatite was deter-
mined by SEM images and XRD analysis on the composite scaffolds’
surface. Results successfully demonstrate that all composite sam-
ples induced apatite formation. While, no hydroxyapatite deposition
was observed on pure alginate scaffold after soaking in SBF. Fig. 6 (c) Alg-0.3BG, 3 days (d) Alg-0.3BG, 21 days
shows some deposits were formed on the composite scaffolds sur-
face, in particular after 21 days immersing in SBF, however, almost
no deposits were observed in pure alginate sample. Furthermore,
they reveal that scaffolds’ bio-mineralization ability highly depends
on the bio-glass content. For example, scaffold samples Alg-1BG and
Alg-1.5BG are significantly more potent than pure alginate scaffold to
form hydroxyapatite. Pure alginate scaffold’s surface was remained
smooth even after 21 days soaking, while a hydroxyapatite rich layer
was found on the surface of Alg/BG composites. It also can be seen
that bio-mineralization increased by increasing the immersing time
from 3 days to 21 days, particularly in sample Alg-0.3BG on which
almost no layer can be observed after 3 days immersing.
Fig. 7 shows the representative XRD spectra of the scaffolds,
which characterizes the evaluation of hydroxylapatite formation.
The XRD spectra of bio-glass and composite scaffolds sample Alg-
(e) Alg-1BG, 3 days (f) Alg-1BG, 21 days
1BG and Alg-1.5BG after immersion in SBF for 3 days showed sharp
peaks at about 32 and 46◦ (2h) which are attributed to (211) and
(222) planes of hydroxyapatite according to [48], while no specific
peak was detected in XRD spectra of alginate and composite sample
Alg-0.3BG scaffold.

3.5. Degradation and dissolution studies

The degradation profile of pure alginate and Alg/BG composite

scaffolds after 60 days of immersion in PBS solution at 37◦ C is shown
in Fig. 8. The degradation rate of scaffolds is critical for their long-
term performance, since it is the basis for following morphogenesis
and bone formation. Initially, by the 7 days, the degradation of the
scaffolds was significantly increased with increase in bio-glass con-
tent. However, as time passed, the pure alginate scaffold exhibited a
faster degradation rate due to its hydrophilic nature and its higher (g) Alg-1.5BG, 3 days (h) Alg-1.5BG, 21 days
swelling ratio as discussed in Section 3.2. Meanwhile, the compos-
ite scaffold sample Alg-0.3BG displayed an increase at a declining Fig. 6. SEM images of scaffold samples surfaces after SBF immersion for 3 and 21 days.
degradarion rate during 60 days. However, the weight of compos-
ite scaffold samples Alg-1BG and Alg-1.5BG, increased slightly after
1264 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267

noted that probably the degradation as observed in in-vitro experi-

ments would not be the same under in-vivo due to the presence of
various enzymes in an in-vivo environment.
(e)
Table 2 shows the concentration of five dissolved inorganic ele-
ment of the PBS solution after 60 days incubation of alginate and
(d) composite scaffolds without refreshing the media. The concentra-
tion of Calcium, Silicate, Phosphorous, Zinc and Magnesium ions
were detected by ICP, while fresh PBS was set as a control. The
intensity (a.u.)

results showed that by increasing the bioglass powder in scaffold,

20 40 60 80
2 in composite scaffolds. The Magnesium concentration increased up
Fig. 7. XRD spectra of in-vitro biomineralization of a) Bioactive glass powders b)
Scaffold sample Alg-1.5BG c) Scaffold sample Alg-1BG d)Scaffold sample Alg-0.3BG e)
Alginate scaffold after 3 days in SBF solution.
2 weeks of incubation in SBF. Thereafter, the degradation rate of
both composite scaffolds increased slightly again. After 60 days of
in-vitro biodegradation, the pure alginate scaffold and composite
scaffold sample Alg-1BG were degraded by 18.5 and 11.8%, respec-
tively. These experimental results indicate that bio-glass particles
used in alginate matrix prevent acceleration of scaffold degradation,
suggesting the formation of HA layer on the surface, making scaf-
fold available for a longer duration for the formation of bone tissue.
Furthermore, dissolution of bio-glass particles buffers the solution at
the polymer surface which slows down the alginate degradation. In
addition, calcium ions existing in PBS solution which were refreshed
every three days as well as the ones created by bio-glass dissolution
are likely lead to cross-linking of alginate matrix which enhances the
scaffold stability and as a result retard its degradation. It should be
20
18
Degradation percentage (%)
the amount of Ca2+ increased to 100.91, respectively, while no PO3−4
was observed. Since the formation of hydroxyl apatite on the com-
(c)
posite scaffolds was proved by SEM and XRD analysis in Section 3.4
and concerning the fact that fresh PBS was set as a control solution,
(b)
the Phosphorous ion concentration in the soaking fluid is likely to
decrease. The SiO4−
4 concentrations of scaffolds increased by increas-
ing the bioglass content, as the apatite formation process did not
consume Si ions. The antibacterial behaviour of the composite scaf-
(a) folds is owed to the Zinc and Magnesium ions release due to the
bioglass degradation. The ICP test results also indicate that Magne-
sium release content significantly exceeded the Zinc release content
100
to 4.22 ppm, while Zinc dissolution concentration was only up to
0.21 ppm. The latter result is aligned with the results of research
conducted in [49]. A meaningful correlation was detected among
the released ions concentrations. Despite the fact that all afore-
mentioned ions are released from bio-glass particles, slight Zn2+
concentration and no PO3− 4 ions was detected in the solution. The
latter could be due to PO3−4 ions tendency to combine with Zn
2+
2+ 2+
and Ca ions [50]. Positive value for Ca concentration in alginate
scaffold was produced by alginate matrix degradation, since it was
crosslinked by Ca2+ ions.
3.6. Antibacterial activities of scaffolds
By determining the bacterial inhibition of all samples after incu-
bation for a fixed time of 12 h, the antibacterial effects of Zn-Mg-
containing composites were evaluated against both gram-negative
(E. coli) and gram-positive (S. aureus) bacteria. As shown in Fig. 9,
significant increase in bacterial inhibition of both S. aureus andE. coli
were observed for Alg-1.5BG compared to alginate scaffolds (34% and
56%, respectively), which proved the antibacterial efficacy of bioglass
containing Zn and Mg. The results showed that E. coli bacteria was
more susceptible to the presence of bioglass than S. aureus bacteria,
which could be due to the cell wall structure differences between
gram-positive and gram-negative bacteria and different sensitivity
of these bacterias to antibacterial elements [51]. In other words,

16 bacterial growth is dependent on both material composition and

bacterial strain type. Although the alginate scaffold showed slight
14 antibacterial efficacy against E. coli andS. aureus, the distinct effect
was observed only after introducing bioglass in composite scaffolds.
12
The results indicate that the bioactive glass containing Zn and Mg
10 played key roles in antibacterial efficacy enhancement of scaffolds.
According to the literature, the released Zn and Mg ions from bioma-
8 terials have shown distinct antibacterial activity [52–55]. The release

6
Alginate scaffold
Alg-0.3BG Table 2
4
Alg-1BG Measurement of different ions (Ca, Si, P, Mg and Zn) level of different scaffolds after
Alg-1.5BG immersing in PBS for 60 days.
2
Sample Elements concentration (ppm)
0
0 10 20 30 40 50 60 Ca Si P Mg Zn

Incubation period (days) Alginate 22.14 ND ND ND ND

Alginate-0.3BG 77.68 45.45 ND 2.17 0.05
Alginate-1BG 96.53 60.38 ND 2.81 0.14
Fig. 8. Degradation rate of pure alginate scaffold as well as Alg/BG composite scaffolds
Alginate-1.5BG 100.91 66.54 ND 4.22 0.21
after soaking in PBS for various periods.
 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267 1265

100 These cells are widely used to test the biocompatibility of scaffolds
Alginate [57–59]. MTT is a colorimetric assay based on cell metabolic activ-
90 Alg-0.3BG ity and the reduction of tetrazolium which has a yellow color to the
Alg-1BG insoluble formazan crystal which is purple. MTT assay was carried
80
Alg-1.5BG out as described in the experimental section. It is also worth noting
Bacterial Inhibitation (%)

70 that increase of Ca2+ ions in culture medium may cause cell apop-
tosis [37]. Considering that, CaCl2 was used as alginate cross-linking
60 * agent in scaffold fabrication procedure, specimens were washed
50 * carefully and immersed 24 h in media prior to MTT assay. None of
the scaffolds exhibited any significant toxicity in regards to MG-63
40 cell line. The results demonstrate even an increase in cell viability
* in MTT assay. It can be seen that in comparison with pure algi-
30 * * nate scaffold, composite scaffolds exhibited considerably higher cell
density. In addition, cell density on TCP is significantly lower than
20 * composite scaffolds, particularly scaffold sample Alg-1BG and Alg-
10 1.5BG, demonstrating the potential applicability of these composite
scaffolds to bone tissue engineering.
0 Highly porous scaffolds are suitable for tissue engineering appli-
S.aureus E.coli
cations as they allow large number of cells to migrate into inner
space as well as providing accessibility to vital nutrients and flu-
Fig. 9. Antibacterial activities of different scaffolds to show the effect of Bioglass con-
taining Zn and Mg concentrations on % bacterial inhibition. *Different from Alginate
ids [60]. Hence, high viability is expected to obtain in high porous
sample (p < 0.05)(n = 3). scaffolds. Higher viability was observed in Alg/BG composite scaf-
folds in comparison with pure alginate scaffold. It may be related
to more suitable pore morphology as mentioned in Section 3.2. The
of the Zn ions from scaffolds causes several bacterial activities inhi- morphology of the cultured cells on the composite scaffolds after 12,
bition such as glycolysis, transmembrane proton translocation and 24 and 72 h incubation were shown in Fig. 11. After initial incuba-
acid tolerance which leads to bacterial disruption [56]. Furthermore, tion period of 12 h, the MG-63 cells were attached in spherical shape.
releasing Mg ions changes the alkalinity of media and create a high While, after 24 h, the cells start to spread on the scaffolds surface. The
osmotic pressure in the bacteria cells which causes bacteria inacti- cells attached and spread more favorably on the composite scaffolds
vation [55]. As shown in Section 3.5, the Zn2+ and Mg2+ ions release containing bioglass particles. As described in Section 3.4, the disso-
increased with increasing bioglass content. As a result, the viability lution of bioglass stimulate the formation of hydroxyapatite on the
of E. coli andS. aureus bacteria were significantly inhibited due to the surface, which is suitable for osteogenic cell attachment and prolifer-
Zn2+ and Mg2+ ions release from composite scaffolds, making them ation [15]. After 72 h of incubation, the cells are completely flattened
a potential candidate for bone tissue engineering. and spread on the composite scaffolds surface, which makes it dif-
ficult to detect cells. individually. The results demonstrate that the
3.7. Cell viability and cell morphology MG-63 cultured cells were well-attached spread and proliferated.

It is known that biological evaluation of biomaterials is an exten-
sive and expensive procedure. However, in order to have insights
to the matter and to avoid in-vivo tests in early stages, it is widely
accepted to perform in-vitro testing before trials involving either ani-
mal or human. Therefore, in this paper in-vitro cell culture method
which represents scaffolds biocompatibility was carried out as fol-
lows. Prepared scaffolds of Alg/BG were subjected to cytotoxicity
using MTT assay with MG-63 cell lines, which are shown in Fig. 10a.
3.8. Alkalin phosphatase assay
ALP activity was tested with MG-63 cells at 7 and 14 days as
an indicator for bone formation and osteoblastic differentiation. As
shown in Fig. 10b, cells cultured on composite scaffolds show higher
ALP activity in comparison with those cultured on pure alginate scaf-
fold. Particularly, the incorporation of bioglass more than 1 g into
alginate polymer promoted the expression of ALP significantly (P <

Alginate

Alg-0.3BG Alginate
0.6 Alg-1BG Alg-0.3BG
30 Alg-1BG
Alg-1.5BG
Alkalin Phosphatase (ng/ml)

Alg-1.5BG
Optical Density

TCP

*
0.4 20
*

0.2 10

0
0.0 7 14
12 24 72 Time (days)

Culture Time (hour)

(a) Cell viability (b) ALP activity

Fig. 10. (a) Cell viability (using MTT assay) and (b) ALP activity of alginate and composite scaffolds using MG-63 cell lines.
1266 D. Zamani, F. Moztarzadeh and D. Bizari / International Journal of Biological Macromolecules 137 (2019) 1256–1267
(a) Alginate, 12h (b) Alg-0.3BG, 12h
(e) Alginate, 24h (f) Alg-0.3BG, 24h
(i) Alginate, 72h (j) Alg-0.3BG, 72h
Fig. 11. SEM images of cell attachment and spreading of MG-63 on alginate and composite scaffolds for 12, 24 and 72 h.
0.05). This result is consistent with the biomineralization results
obtained in Section 3.4. There are two possible explanations. One is
that the formation of apatite may improve the MG-63 cell response
to the scaffolds and cell attachment. As a result, the higher ALP activ-
ity was observed [4]. The other one might be attributed to various
ion release from bioactive glass particles that might activate gene
expression in osteoprogenitor cells [37]. The MG-63 cells seeded on
composite scaffolds exhibited higher ALP expression at 14 days in
comparison with 7 days.
4. Conclusion
We demonstrated that with the incorporation of bio-glass con-
taining Zinc and Magnesium could easily improve the mechanical
properties, the antibacterial efficacy and the bioactivity of alginate
scaffolds. Despite the proven positive results of doping Zinc and
Magnesium ions, to the best of authors knowledge, these are the
first composite scaffolds that include bio-glass particles containing
Zn and Mg for bone tissue engineering applications. In this study,
alginate, a nontoxic, biodegradable, natural polymer was used as
matrix of the composites. Bio-glass particles, fabricated by sol-gel
technique, distributed uniformly in alginate matrix with almost no
agglomerates. The increase in bio-glass amount in relation to algi-
nate polymer leads to decrease in pore size. Significant improvement
in mechanical properties and degradation rate as well as HA forma-
tion after incubation of obtained composites in SBF, which can be
tailored by changing the concentration of bio-glass particles, proved
the advantages of Alg/BG composites over pure alginate scaffolds.
(c) Alg-1BG, 12h (d) Alg-1.5BG, 12h
(g) Alg-1BG, 24h (h) Alg-1.5BG, 24h
(k) Alg-1BG, 72h (l) Alg-1.5BG, 72h
Moreover, in this paper, the results of in-vitro tests were presented
which was promising as the Alg/BG composites exhibited good
MG-63 cell response (viability, attachment and proliferation) and
osteoblast differentiation. ICP analysis results after PBS incubation
of the obtained composites show the composites ion release. The
measurement of antibacterial activity indicated that these released
ions restrict the growth of both S. aureus andE. coli. Thus, it can be
concluded that the alginate bio-glass composites can be a good can-
didate for bone tissue engineering. In order to further the biological
evaluations, the in-vivo tests are currently being carried out and the
results will be published in future as the ongoing work of this paper.
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