1. Evaluation of zinc and magnesium doped 45S5 mesoporous bioactive
glass system for the growth of hydroxyl apatite layer
Vikas Anand, K.J. Singh ⁎, Kulwinder Kaur
Department of Physics, Guru Nanak Dev University, Amritsar 143005, India
a b s t r a c ta r t i c l e i n f o
Article history:
Received 9 July 2014
Received in revised form 21 September 2014
Accepted 28 September 2014
Available online xxxx
Keywords:
Mesoporous bioactive glass;
Tris SBF;
Apatite layer
Bioactive glass samples of xZnO(22.4 − x)Na2O·46.1SiO2·26.9CaO.2·6P2O5·2MgO system have been prepared
by using the sol gel technique. Investigations of structural and bioactive properties of these samples have been
undertaken by using X-ray diffraction, field emission scanning electron microscopy, Raman and energy-
dispersive X-ray spectroscopy, Brunauer, Emmett and Teller technique, thermogravimetry, differential thermal
analysis and derivative thermogravimetry methods and pH studies. X-ray diffraction spectra of prepared samples
indicate the formation of hydroxyl apatite layer after 7 and 14 days during in vitro analysis. Observed peaks of
Raman spectra confirm the growth of hydroxyl apatite layer during in vitro analysis. The formation of apatite
phase is responsible for change in the morphology of samples which has been studied by field emission scanning
electron microscopy. Ca/P ratio and ion dissolution rate have been evaluated from energy dispersive X-ray spec-
troscopy results. The observed Ca/P ratio further confirms the growth of apatite layer with time during in vitro
analysis. The effect of the addition of zinc on surface area and mesoporous nature of all the samples has been
investigated by the Brunauer, Emmett and Teller technique. All glass samples have been found to be thermally
stable from thermogravimetry, differential thermal analysis and derivative thermogravimetry techniques. pH
studies indicate the non-acidic nature of all the prepared glass samples. Drug release properties of the glass sam-
ples have been investigated by using gentamycin as an antibiotic. Prepared samples have shown excellent drug
release properties which can be related to the porous nature of the samples. Due to slow dissolution rate, good
drug delivery properties and formation of apatite phase within 7 to 14 days, our glass samples can be promising
candidates for bone regeneration applications.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Fracture in the bone may cause a serious problem when its impact is
on a large scale. Sometimes, fracture can lead to the loss of the bone and
there can be many other incidents like cancer therapies, accidents, and
age factors (old age) responsible for the elimination of the bone from
the human body. In a survey, it has been estimated that in women
and men (specially over the age of 50 years), the lifetime risk of devel-
oping a fracture is 40% and 13% respectively [1]. Replacement of dam-
aged bone is a major problem in orthopedics surgery and in order to
meet this purpose, bioinert metallic implants are used. These implants
act like a foreign particle for host body and sometimes, they may get
rejection. Moreover, metallic implants may not stimulate the osteoblas-
tic and osteoconductive phenomena in the host body. Due to metallic
nature, these implants are temperature sensitive and start to cause the
pain on surrounding tissues under hot and cold conditions. To overcome
all these problems, a special kind of glass has been developed which has
the ability to bond with bone and soft tissues [2]. This glass is popularly
known as ‘bioactive glass’. Bioactive glasses are one of the categories of
bio-materials and they can be potential materials for applications in
orthopedics and surgery. Bioactive glasses are bioresorbable and they
provide friendly environment for the growth of bone (apatite layer)
and soft tissues. Therefore, these can be good alternatives of metallic
implants. First, bio-active glass was prepared by using the conventional
melt-quenching technique by Larry Hench. This glass was termed as
45S5 Bioglass®. Lower Ca/P ratios do not bond to bone [3]. Bioactive
glasses can also be prepared by the sol–gel technique.
Recently, borate glasses have also been investigated for biomedical ap-
plications by removing silica from the bioactive glass compositions [4,5].
Silica free glass samples with the composition (45B2O3, 24.5Na2O,
24.5CaO, 6P2O5) were prepared with additional 2% of fluoride cations
which were added separately. It has been observed that dissolution rate
is better for LiF as compared to other fluorides (ZnF2, CaF2 and NaF) [4].
Oxide glass samples of the B2O3, Na2O, CaO, K2O and LiO constituents
have also been prepared in the laboratory [5]. It has been noticed that
dissolution rate proceeds rapidly than as observed in silicate Hench's
bioglass (45S5). It has been inferred that the corrosion mechanisms of
borate glasses in aqueous environments, generally undergo hydration,
hydrolysis, and ion exchange reactions.
Journal of Non-Crystalline Solids 406 (2014) 88–94
⁎ Corresponding author.
E-mail address: kanwarjitsingh@yahoo.com (K.J. Singh).
http://dx.doi.org/10.1016/j.jnoncrysol.2014.09.050
0022-3093/© 2014 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of Non-Crystalline Solids
journal homepage: www.elsevier.com/ locate/ jnoncrysol

2. In order to check the bioactivity of the samples, the following is the
conventional procedure. Samples are soaked in the TRIS Simulated Body
Fluid (SBF). SBF has concentration of ions equal to human blood plasma.
The creation of apatite layer on the samples (from few hours up to few
days) can confirm the bioactive nature of prepared samples. It has been
found that formation rate of apatite layer improves at higher concentra-
tion of calcium ions when the phosphate ions remain in the range of 4–
6 mol% in the composition. When the composition of phosphate ions is
increased from 6 mol% onwards, it shows a negative effect in the rate of
formation of apatite layer.
Applications of bioactive glass in human body depend upon its bio-
activity, structural and dissolution properties. Bioactivity of glass pro-
vides the information regarding the growth of apatite layer, whereas,
structural and dissolution rate properties inform about the strength of
bioactive glass. 45S5 has been the most successful bioactive glass. More-
over, 45S5 bio-glass cannot be used as a good scaffold owing to high
dissolution rate. In this work, authors have made an attempt to improve
the dissolution rate of 45S5 bio-glass along with constant Ca and P ratio.
All glass samples have been prepared in the laboratory by the sol gel
technique.
2. Materials and method
Glass system xZnO(22.4 − x)Na2O·46.1SiO2·26.9CaO.2·6P2O5·
2MgO was prepared in the laboratory by using the sol gel method.
Details of the chemical composition of the glass samples are provided in
Table 1. The solutions to obtain glasses were prepared from the stoichio-
metric amounts of tetraethyl orthosilicate (TEOS), triethyl phosphate
(TEP), Ca(NO3)2·4H2O, NaNO3, Mg(NO3)·6H2O and Zn(NO3)·4H2O (AR
grade). 1 M HNO3 was used as the catalyst for hydrolysis process.
TEOS was added into 1 M HNO3 solution (TEOS and H2O molar ratio
equal to eight) and the mixture was stirred up to 1 h for complete
hydrolysis. TEP, calcium nitrate tetra hydrate and magnesium nitrate
hexa hydrate were dissolved in 1 M HNO3 solution and stirred up to
more than 40 min. Both solutions were mixed under vigorous stirring
and sodium nitrate was added into the solution. After 1 h of vigorous
stirring, transparent solution was obtained. Solution was kept in an air
tight beaker for 5 days for aging. Gel was heated up to 60 °C for 12 h,
120 °C for 12 h and the product had been calcinated up to 700 °C for
4 h. Prepared samples had been crushed in agar and mortar for 1 h.
X-ray diffraction (XRD) study has been undertaken by using BRUKER
D8 FOCUS XRD machine. Raman studies of prepared samples have been
performed by the RENISHAW IN VIA REFLEX MICRO RAMAN SPEC-
TROMETER with 785 nm laser beam of exposure time of 40 s with the
range of 300 to 1500 cm−1
. Field emission scanning electron microsco-
py (FESEM) study has been carried out by ZEISS SUPERA 55. In order to
get FESEM images, samples have been filtered from SBF and washed
with acetone and DI water four times. Moisture has been removed
from samples by drying them up to 60 °C. Platinum coating has been
used to make the samples conductive. Gold coating of samples has
been avoided due to overlapping of gold and phosphorus peaks during
energy dispersive X-ray (EDX) analysis. Brunauer–Emmett–Teller
(BET) analysis has been undertaken by micrometrics ASAP 2020.
Thermogravimetry, differential thermal analysis and derivative
thermogravimetry (TG–DTA–DTG) techniques have been used to inves-
tigate the thermal behavior of prepared bioactive glasses by EXSTAR TG/
DTA 6300 instrument up to 1400 °C with the increase in temperature of
10 °C per minute.
3. Assessment of in vitro bioactivity
Bioactivity nature of samples had been evaluated with the help of
27-Tris SBF solution. SBF solution was prepared as per the recipe report-
ed elsewhere [6]. 3 mg of powder sample was soaked in 30 ml of 27-Tris
SBF solution for 7 and 14 days under 37 °C temperature. After every
12 h, sample was replaced by a fresh TRIS SBF solution. pH of Tris SBF
was measured after regular interval of time to check the ion exchange
process in between the surface of sample and Tris SBF solution. Ion con-
centration of Tris SBF and human blood plasma is provided in Table 2.
The results of in vitro bioactivity were analyzed with the help of XRD,
Raman, SEM and EDX studies.
4. Results and discussion
4.1. XRD studies
XRD patterns of the samples are provided in Fig. 1. All samples show
amorphous nature of material before soaking into SBF. As shown in the
Fig., sharp peaks of Calcium Hydroxide Phosphate [Hydroxylapatite
(HAp)] (JCPDS No. 00-074-0566) are observed after 7 days. Intensity
of peaks has increased when the number of days is increased to
14 days. Along with HAp layer, calcium carbonate and calcium phos-
phate peaks are also observed in all three XRD patterns. The formation
of HAp layer on the surface of prepared glass samples and shifting of
amorphous to crystalline behavior after soaking of glass samples in
SBF solution with the passage of time indicate the bioactive behavior
of prepared samples. Sometimes, there may be slight shifting of peaks
of HAp due to the small substitution of calcium magnesium in biological
apatite [7].
4.2. Raman spectroscopy
It is shown in the Raman spectra that before the in vitro analysis, one
sharp peak appears around 1070 cm−1
indicating the presence of car-
bonate ions (Fig. 2). Same peak splits into two small peaks after 7 and
14 days during in vitro test which indicate the presence of phosphate
ions. Peaks around 1089–1091 and 1052–1055 cm−1
in the Raman
spectra corresponds to the asymmetric stretching (ν3) of the PO
bond in phosphate. The peak around 963–965 cm−1
corresponds to
the symmetric stretching mode (ν1) of the PO bond of the phosphate
group. This peak is generally observed around 963 cm−1
in hydroxylap-
atite [8]. The minor shift from 963 cm−1
may be due to minor incorpo-
ration of magnesium or zinc ions into the HAp lattice. The resultant
compressive stresses in the lattice may lead to this difference in peak
position [9]. Raman data support the inferences of XRD studies in
terms of observation of bioactive nature of the glass samples.
4.3. FESEM and EDX studies
Morphology of samples has been confirmed by FESEM before and
after the immersion of samples in SBF solution. FESEM micrographs
have observed that after 7 days, the content of calcium and phosphate
Table 1
Composition (in mol%) of samples.
Sample code ZnO Na2O SiO2 CaO P2O5 MgO
BG-1 6 16.4 46.1 26.9 2.6 2
BG-2 8 14.4 46.1 26.9 2.6 2
BG-3 10 12.4 46.1 26.9 2.6 2
Table 2
Ion concentration of Tris SBF and Human plasma.
S. no. Ions Ion conc. (in 27-Tris SBF) Ion conc. (in human plasma)
1 Na+
142 142
2 K+
5 5
3 Mg2+
1.5 1.5
4 Ca2+
2.5 2.5
5 HPO4
2−
1 1
6 HCO3
−
27 27
7 Cl−
125 103
8 SO4
2−
0.5 0.5
89V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

3. starts to increase which indicates that the apatite layer (calcium phos-
phate) starts to generate on the surface of samples (shown in Fig. 3)
After 14 days, growth of layer has been observed to increase and almost
the whole surface of the sample has been found to be covered by the ap-
atite layer. This growth phenomenon is common in all three prepared
samples but the growth rate of the layer is high at the surface of BG-1
and low at the surface of BG-3. It may be due to two reasons:
(i) Increase in ZnO content (BG-3 sample). The presence of ZnO or
MgO in the samples may have slowed down the deposition rate of
HAp. This observation is consistent with the results of Li et al. [10] and
Du and Chang [11]; and (ii) lesser surface area for growth of apatite
layer (BG-3 sample, Table 4).
Due to small surface area in BG-3 than BG-1, the rate of growth of ap-
atite layer may have slowed down. It is reported by Li et al. that leaching
of ions is one of the reasons to slow down the rate of apatite layer but
when zinc is added in the presence of magnesium with the replacement
sodium content, there is a decrease in the leaching of Mg, Zn and silicon
ions as shown in EDX (Fig. 4). This can be explained as follows. Both Mg
and Zn ions have a dual character to behave like a network former or
network modifier. If they act like a former then SiOSi bond is
replaced by SiOMg or SiOZn bond and as a result, structure
becomes weak because the newly formed bond has less strength as
compared to the old one. But in case, Mg and Zn behave like modifiers
they give strength to the structure which reduces the solubility of
glass in SBF [12].
Enhancement in growth of apatite layer may be due to the presence
of magnesium ions along with zinc ions. Magnesium being a major ele-
ment in the bone, may increase the growth of apatite layer. Moreover,
magnesium plays a vital role in providing the apoptosis control to cell.
EDX study of the prepared samples has been undertaken before and
after 7 and 14 days of immersion in SBF. In EDX study, it has been
Fig. 1. XRD pattern of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after the
in vitro analysis.
Fig. 2. Raman peaks of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after the
in vitro analysis.
90 V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

4. observed that there is an increase in the concentration of calcium and
phosphorus with time due to the formation of apatite layer on the
surface of the sample but the dissolution of silicon ions is very slow in
all three samples. Due to this low solubility rate, these samples may
show good scaffold properties. Exchange of ions is also confirmed by
EDX graphs (Fig. 4). The graphs show the change of calcium and phos-
phorus ions from Tris SBF and the growth of apatite layer on the surface
of glass has been established within seven days.
At the end of 14th day, the concentration of calcium and phosphorus
ions increases due to the formation of calcium phosphate phases on the
surface of glass. Phase formation has also been confirmed with the
weight (%) data of EDX (Table 3). The results indicate that Ca/P ratio
of samples changes before and after in vitro analysis. Ca/P ratio has
been reported as 1.67 for the stoichiometric composition of HAp [13].
Synthetic calcium phosphate materials have also been prepared and
studied extensively in vitro and in vivo. Some materials have been called
“hydroxylapatite” with Ca/P ratios ranging from 1.3 up to 2.0 [14]. Dur-
ing the EDX analysis for our glass samples, authors have found that after
14 days, Ca/P ratio lies in the range of 1.68–1.77 for all the glass samples
prepared in the laboratory. These values are very close to Ca/P ratio
(1.67) for stoichiometric composition of HAp and also, lie in the range
of already reported values of Ca/P ratio (1.3–2.0) for hydroxyl apatite.
These results confirm the growth of HAp on the surface of the glass sam-
ples. Moreover, these results also support the inferences of XRD and
Raman studies. No major change in the concentration of Si, Mg and Zn
ions has been observed even after 14 days of samples in Tris SBF
solution.
4.4. Brunauer–Emmett–Teller studies
BET study has been undertaken to find out the surface area and
porous nature of prepared samples (Fig. 5). N2 adsorption–desorption
phenomena have been studied to obtain the desired results. BET surface
area is calculated by using all adsorption data points from .01 to 1.0
(total points 46) in the relative pressure (P/Po). In order to calculate
the pore size, the Barrett–Joyner–Halenda (BJH) desorption method
(relative pressure from 1.0 to .12, total 16 points) has been preferred
over the adsorption method because desorption is carried out at a low
relative pressure which is useful for thermodynamic equilibrium [15].
Passage of time indicates the bioactive behavior of prepared samples.
Sometimes, there may be slight shifting of peaks of HAp due to small
substitution of calcium magnesium in biological apatite [7]. Surface
area, pore volume and BJH desorption average pore volume of prepared
samples are provided in Table 4. As shown in the table, pore volume and
pore size of samples decrease with the increase of the amount of Zn.
This is due to the role of Zn as the network modifier to increase the
strength of glass samples.
Fig. 5 shows the N2 physisorption i.e. adsorption and desorption
isotherms and pore size distribution of prepared bioactive samples.
The pore size of samples (Table 4) comes in the range of type II
(2–50 nm). According to International Union of Pure and Applied
Chemistry (IUPAC) classification of adsorption isotherm, type II de-
scribes the presence of mesoporous and microspores which exhibits
a hysteresis loop and a variation point at lower pressure in a material
[16].
Fig. 3. SEM images of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after in vitro study.
91V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

5. 4.5. TG/DTA/DTG studies
Graphical representation of thermal behavior of samples is given
in Fig. 6. There is no sharp change in the mass of samples. For BG-1,
BG-2 and BG-3, there is 5.60%, 5.24% and 2.6% loss of weight up to
300 °C respectively which is due to the evaporation of adsorbed
water and ethanol molecules. BG-1 has high porosity which may be
a reason of highest weight loss among all three samples. After that,
from 300 to 700 °C, the weight loss is 2.81%, 1.86% and 2.09% for
BG-1, BG-2 and BG-3 respectively. This may be due to the decompo-
sition of precursor and removal of nitrate from samples. Further,
samples are heated up to 1400 °C with 0.57%, 0.72%, and 0.79%
weight loss of BG-1, BG-2 and BG-3 respectively. Weight loss from
(700 to 1400 °C) may be due to the decomposition of carbonate
and formation of crystalline phases in the sample. All three samples
show a stable thermal response of up to 1400 °C.
4.6. pH studies
pH study of samples gives an idea about ion exchange process in be-
tween the surface of sample and Tris SBF solution. Variation of pH of
samples in Tris SBF is shown in Fig. 7. A sudden rise in the pH of solution
indicates the exchange of ions between sample and Tris SBF which
started in the first hour. Total variation of pH of solution is from 7.4 up
to 8.74 within 180 h. The pH of solution changes in a narrow range of
1.34 which shows that samples are chemically stable in Tris SBF solution
and do not show any acidic nature in Tris SBF.
Fig. 4. EDX results of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after in vitro study.
Table 3
Weight percentage of the elements of samples from EDX data. EDX results are average (Avg.) of 5 measurements and the observed error (Er) percentage is also presented.
Element Standard used BG-1 (wt.%) BG-2 (wt.%) BG-3 (wt.%)
Before After 14 days Before After 14 days Before After 14 days
Avg. ±Er%x10 Avg. ±Er%x10 Avg. ±Er%x10 Avg. ±Er%x10 Avg. ±Er%x10 Avg. ±Er%x10
C CaCO3 14.6 0.02 16.44 0.024 14.45 0.031 16.44 0.01 9.62 0.031 13.01 0.039
O SiO2 48.59 0.03 47.02 0.032 49.72 0.034 49.11 0.04 48.35 0.041 49.01 0.021
Na Albite 12.97 0.025 8.48 0.031 8.48 0.023 6.48 0.028 2.63 0.031 1.13 0.041
Mg MgO 2.78 0.031 3.28 0.034 3.28 0.025 3.28 0.032 0.54 0.029 0.41 0.031
Si SiO2 14.65 0.032 14.12 0.031 14.12 0.027 13.12 0.031 23.51 0.028 18.47 0.021
P GaP 1.96 0.031 2.51 0.032 2.68 0.041 3.12 0.049 4.54 0.05 4.98 0.043
Ca Wollastonite 2.11 0.021 4.21 0.041 3.13 0.048 5.31 0.042 4.92 0.031 8.8 0.029
Zn Zn 2.34 0.021 4.14 0.01 4.14 0.014 3.14 0.021 5.89 0.018 4.23 0.022
Ca/P ratio 1.07 1.68 1.17 1.7 1.08 1.77
92 V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

6. 4.7. In vitro drug release studies
In vitro drug release of sample has been studied with the help of
gentamicin. 2 g of prepared sample has been immersed in 40 ml of
gentamicin solution (10 mg ml−1
). Sample has been kept in the solution
up to 24 h. After filtering the powder and drying at 40 °C up to 48 h,
in vitro release of gentamicin from the drug-loaded bioactive glass is
carried out in an incubator at 37 °C. 2 g of powder is dipped in the
20 ml of Tris SBF under 37 °C. Gentamicin release was determined by
UV analysis. The release medium was withdrawn at the predetermined
time intervals and replaced with fresh SBF solution each time. During
the drug release mechanism, all three samples show a quick release in
the first hour and then there is a decrease in the rate of release of drug
in SBF (shown in Fig. 8). All the three samples show similar drug release
behavior as reported by mesoporous channel [17] This study shows that
prepared samples have a good response in drug delivery phenomena
and it is due to their mesoporous behavior.
4.8. Statistical calculations
Data for pH and drug delivery studies is the average of 3 times rep-
lication of experiment. Error bars have been used in the corresponding
figures. EDX measurements have been undertaken 5 times. The ob-
served maximum deviation is 0.5%.
5. Conclusions
XRD peaks at 25.8°, 28.9°, 31.7°, 32.7°, 32.19°, 32.8°, and 39.78° indi-
cate the presence of HAp phase on the surface of samples during in vitro
analysis. Growth of HAp phase has been confirmed by HAp(PO bond)
peak at 963 cm−1
in Raman spectra. FESEM images further confirm the
findings of XRD and Raman investigations. Observed Ca/P ratio (1.68,
1.70 and 1.77) by the EDX technique during in vitro analysis also con-
firms the formation of HAp phase. Low dissolution rate of silicon, mag-
nesium and zinc ions has been observed by the EDX analysis. From BET
study, it can be inferred that the increase in the content of zinc leads to
the decrease in the surface area and pore size which can be related to
the network modifier behavior of zinc. These results are consistent
with the findings of the TGA–DTA–DTG technique which shows a stable
thermal behavior of samples having high content of zinc. Drug release
properties concluded that glass sample 6ZnO·16.4Na2O·46.1SiO2·
26.9CaO·2.6P2O5·2MgO is better due its higher surface area and pore
size. Porosity, surface area and chemical composition of the bioactive
glasses have been found to play a significant role in controlling the bio-
active behavior of the glass samples.
Fig. 5. (a) Adsorption desorption curves and (b) pore volume of bioactive samples. Lines have been drawn as a guide to the eye.
Table 4
BET surface area, pore volume and pore size of bioactive samples.
Sample code BET surface area (m2
/g) Pore volume (cm3
/g A°) Pore size (nm)
BG-1 14.9 7.5 × 10−4
30
BG-2 13.8 3.8 × 10−4
21
BG-3 9.4 1.4 × 10−4
18
Fig. 6. TG–DTA–DTG response of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples.
93V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

7. Acknowledgments
The authors Vikas Anand and Kulwinder Kaur are grateful to the
financial assistance provided by the UGC, New Delhi (India) through
JRF (NET)[F.17-74/2008 (SA-I)] and DST, New Delhi (India) through IN-
SPIRE program [IF-120620] respectively.
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Fig. 7. pH variation of samples during in vitro analysis. Lines have been drawn as a guide to
the eye.
Fig. 8. Gentamicin release by glass samples after regular time intervals. Lines have been
drawn as a guide to the eye.
94 V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94