This document discusses the development of bioactive ceramics for bone regeneration applications. The ceramics were developed in the B2O3-MgO-SiO2-Na2O-CaO-P2O5-ZnO system using a sol-gel technique. Five compositions were prepared with varying amounts of B2O3. Characterization showed the materials were mesoporous and crystallized phases including hydroxylapatite and whitlockite. In vitro tests found the materials degraded appropriately and formed hydroxylapatite layers when soaked in simulated body fluid, indicating their bioactive nature. Biological studies of swelling, drug release, cytotoxicity and cell culture were also conducted. The results suggest these materials are promising

1. CERAMICS
INTERNATIONAL
Available online at www.sciencedirect.com
Ceramics International 42 (2016) 3638–3651
B2O3–MgO–SiO2–Na2O–CaO–P2O5–ZnO bioactive system for bone
regeneration applications
Vikas Ananda
, K.J. Singha,n
, Kulwinder Kaura
, Harpreet Kaurb
, Daljit Singh Arorab
a
Department of Physics, Guru Nanak Dev University, Amritsar 143005, India
b
Department of Microbiology, Guru Nanak Dev University, Amritsar 143005,India
Received 22 August 2015; received in revised form 22 October 2015; accepted 4 November 2015
Available online 12 November 2015
Abstract
Bioactive samples of composition x Á B2O3 Á (2xþ2)MgO.(22.4À(2xþ2))Na2O.(46.1Àx) SiO2.26.9CaO Á 2.6P2O5 Á 2ZnO (x varying from 0 to
4) have been prepared in the laboratory by the sol–gel technique. Structural information has been drawn from X-ray Diffraction, Fourier
Transform Infrared spectroscopy, Field Emission Scanning Electron Microscopy, Energy Dispersive X-ray and Atomic Absorption Spectroscopy.
By using the Brunauer, Emmett and Teller technique, it has been found that the samples are mesoporus with pore size varying from 19 to 42 nm.
Detailed analysis of degradation behavior of the materials has been undertaken. Gentamycin has been tested as an antibiotic to study their drug
release properties. Swelling, antimicrobial and cell culture studies have also been conducted. Attempt has been made to search for suitable
chemical composition for the purpose of developing effective implant material for bone regeneration applications.
& 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Keywords: Bioceramic; SBF; Hydroxylapatite
1. Introduction
Hard tissue replacement or regeneration are the only clinical
solutions available for the treatment of broken bone [1]. Due to
bone formation ability, many bioactive glasses or ceramics
have been tested for hard tissue engineering [2]. Several
bioactive systems have been reported but most of them suffer
from several drawbacks like abrupt dissolution rate, slow
bioactivity, poor mechanical strength and lack of some special
properties, for example, antimicrobial, cell proliferation and
osteogenesis, which restrict their use as implant materials for
clinical applications. It has been reported that dissolution rate,
bioactivity and mechanical properties of the bioactive samples
can be improved by addition of different phases including
hydroxylapatite (HAp) and whitlockite [3–5]. Growth of HAp
and whitlockite layers on the bioactive samples are key
indicators for applications of samples as implant materials
for bone regeneration applications. HAp (Ca10(PO4)6(OH)2)
has similar chemical composition to human bone. Mechanical
strength, dissolution rate and osteoblast properties of HAp are
also comparable to human bone. Moreover, HAp can make
strong and stable bond with old bone and the surrounding
tissues. Whitlockite is a term for the mineral or synthetic
material in which Mg2 þ
and HPO4
2À
ions substitute in β-Tri
Calcium Phosphate (TCP). Its solubility is lower than pure β
-TCP which indicate the enhanced stability of the lattice [6]. It
occurs in various pathological calcifications and also, as a
major constituent of human dental calculus [7].
Significant efforts have been made by researchers to
enhance the properties of bioactive samples by doping several
elements like, Ag, Mg, Sr, Zn etc [8–12]. Each dopant can
alter the characteristics which further leads to change in the
properties of bioactive sample. Zinc, magnesium and boron as
constituents of bioactive system have special place due to the
following observations. It has been observed that partial
substitution of zinc with the replacement of sodium in 45S5
bioglasss
improves the bond formation activity by increasing
cell proliferation and differentiation and increased amount of
zinc leads to formation of bone structure [13,14]. Moreover,
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E-mail address: kanwarjitsingh@yahoo.com (K.J. Singh).

2. zinc also provide DNA replication [15], bone re-sorption [16]
and antibacterial properties [16,17]. Zinc helps in the bond
formation in the implant and accelerate the recovery of patient
[14]. Magnesium is one of the most abundant cations in the
human body. As a bone constituent, it covers 50–60% weight
of bone [18,19]. Mg is essential for many enzyme reactions.
Mg can directly stimulate the osteoblast proliferation [20].
MgO also incorporate the synthesis of HAp layer as reported
by Vallet-Regi et al. [1] and Balamurgan et al. [8,21].
Magnesium is naturally present in human bone and essential
for the human metabolism [18,19]. It can stimulate the growth
of new bone and tissues [20,22]. Borate can also be used in
bioactive systems as a dopant. Borate is a very interesting
material for doping because it can easily change its coordina-
tion number from three to four and hence, it can form the
variable structural units [23]. Recently, silica free borate
glasses [24] have also been investigated for biomedical
applications. It has been inferred that the corrosion mechan-
isms of borate glasses in aqueous environments, generally
undergo hydration, hydrolysis, and ion exchange reactions.
Most of studies reported earlier provide information about the
effect of doping of individual ZnO, MgO and B2O3 on the
properties of bioactive ceramics. Four out of five samples
reported in this article contain all the three compounds. The
main aim of presented work is to investigate the effect of
borate and magnesium oxide in the presence of zinc oxide on
HAp and whitlockite layer formation and biological properties.
2. Materials and methods
2.1. Preparation of bioactive samples
Bioactive ceramics of the system x Á B2O3(2xþ2)MgO Á
(22.4À2xþ2)Na2O Á (46.1Àx) SiO2 Á 26.9CaO Á 2.6P2O5 Á
2ZnO have been prepared in the laboratory by using the
sol–gel method. Tetraethyl orthosilicate (TEOS), triethyl
phosphate (TEP), calcium nitrate tetra hydrate, sodium
nitrate, magnesium nitrate hexahydrate, zinc nitrate tetra
hydrate and boric acid (AR grade) have been used as
source materials for SiO2, P2O5, CaO, Na2O, MgO, ZnO
and B2O3 respectively. 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 one hour for complete hydrolysis.
TEP,calcium nitrate tetra hydrate and magnesium nitrate
hexahydrate were dissolved in 1 M HNO3 solution and
stirred up to 40 min. Both solutions were mixed under
vigorous stirring condition. Sodium nitrate was added into
the solution followed by boric acid. After one hour of
vigorous stirring, transparent solution was obtained. Solu-
tion was kept in air tight beaker for 3 days for aging. Gel
was heated up to 60 1C for 12 h and 120 1C for 12 h. The
samples were calcinated up to 700 1C for 8 h to attain
crystalline nature. Prepared samples had been crushed in
agar and mortar for one hour. Chemical composition of the
prepared samples is provided in Table 1.
2.2. Assessment of in vitro bioactivity
In vitro bioactive nature of samples has been evaluated with
the help of simulated body fluid (SBF) solution. SBF solution
has been prepared as per the recipe reported elsewhere [25].
One gram of powder sample was soaked in 50 ml of SBF
solution under 37 1C. After every 12 h, old SBF was replaced
with fresh SBF solution.
2.3. Characterization techniques
X-ray diffraction (XRD) study has been undertaken by using
Bruker D8 focus XRD machine. Fourier Transform Infrared
Spectroscopy (FTIR) investigations of the samples have been
undertaken in transmittance mode by using Perkin Elmer
Spectrometer(C92035),Germany. Field emission scanning
electron microscopy (FESEM) and energy dispersive X-ray
(EDX) studies have 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 1C. Platinum coating has been used to make the samples
conductive. EDX study has been undertaken without coating
of samples. Differential thermal analysis technique has been
used to investigate the thermal behavior of the samples by
EXSTAR TG/DTA 6300 instrument up to 1400 1C with the
increase in temperature of 10 1C minÀ1
. Atomic Absorption
Spectroscopy (AAS) study has been undertaken by using AAS
240FS Agilent Atomic Absorption Spectrometer to check the
concentration of ions in decant SBF and citric buffer.
Biological properties of samples have been studied with the
help of swelling, drug release, cytotoxicity and cell culture
studies. Labsystem Multiskan EX ELISA and Biorad 680-XR,
Japan reader with 570 and 590 nm wavelengths of UV–visible
range have been used for biological studies.
3. Results and discussion
3.1. Apatite forming ability of the system
Apatite formation ability of samples have been checked
before and after immersion in the SBF solution. During these
investigations, structural changes, morphology and Ca/P ratios
have been investigated with the help of XRD, FTIR, FESEM
Table 1
Composition in mol% and pore size in nm. Pore size is the average of
3 measurements and the observed standard deviation (SD) is presented.
Sample
code
B2O3 MgO Na2O SiO2 CaO P2O5 ZnO Pore Size
(nm)7SD.
MZB-0 0 2 20.4 46.1 26.9 2.6 2 4271
MZB-1 1 4 18.4 45.1 26.9 2.6 2 3472
MZB-2 2 6 16.4 44.1 26.9 2.6 2 2571
MZB-3 3 8 14.4 43.1 26.9 2.6 2 1971
MZB-4 4 10 12.4 42.1 26.9 2.6 2 1972
V. Anand et al. / Ceramics International 42 (2016) 3638–3651 3639

3. and EDX studies. The results obtained have been correlated
with the bioactive nature of the samples.
3.1.1. XRD studies
XRD patterns of the samples are provided in Fig. 1.
Sharp peaks of sodium calcium silicate (JCPDF-78-1650)
(in Fig. 1(a)) whitlockite (JCPDF-70-2064) (in Fig. 1(b)–
(e)) along with the appearance of sodium nitrate (Fig. 1
(b)), magnesium phosphate (Fig. 1(e)) and calcite (Fig. 1
(b)–(e)) indicates the crystalline behavior samples. It has
been noticed that peaks of whitlockite phase become
sharper with the addition of magnesium. Magnesium
containing whitlockite is one of the abundant bio-mineral
in the bone [26,27]. However, its importance and role has
not been fully identified. Along with calcium ion, magne-
sium ions are also present in the human bone [26,28].
Dentin contains 26% to 58% of whitlockite mineral by
weight (Fig. 2).
Fig. 1. XRD spectra of sample before and after in vitro analysis of samples (□ Calcite, ∎ Whitlockite  Magnesium phosphate, ▾ Hydroxylapatite, △Sodium
calcium silicate).
V. Anand et al. / Ceramics International 42 (2016) 3638–36513640

4. It is difficult to synthesize the whitlockite phase in pure form
and there are only limited reports available for the synthesis of
whitlockite in aqueous solutions [28–30]. It is also reported
that whitlockite phase is stable only in acidic pH(4.5–5) [31].
In the light of this situation, it is imperative that when our
samples (containing whitlockite phase) come in contact with
high pH SBF solution (pH$7.4), it leads to dissolving of the
whitlockite phase and formation of HAp phase (JCPDF no.
Fig. 2. FTIR spectra of sample before and after in vitro analysis.
V. Anand et al. / Ceramics International 42 (2016) 3638–3651 3641

5. 07-0747) takes place. During immersion of samples in SBF,
structural changes have been observed after 2, 7 and 14 days.
Within 2 days, it has been seen that due to exchange of ions
between samples and SBF, the pH of solution rose up to 8.4. It
has also been seen that calcite phase (in Fig. 1(b)–(e)) start to
form with the exchange of calcium ions between sample and
SBF solution. After few hours, calcium carbonate dissociate
into calcium and carbonate ions leading to higher concentra-
tion of Ca2þ
ions in the solution which can be used for the
formation of apatite layer. Reaction may occur as per the
following equation;
CaCO3 þHþ
Ca2 þ
þHCOÀ
3 ð1Þ
After every 12 h, old SBF has been replaced with fresh SBF
solution to maintain the ions concentration and pH of solution.
Within the 7days, stable HAp phase start to grow on the
surface of samples. Peak at 31.91 in the XRD spectra indicates
the formation HAp phase after 7 days (Fig. 1(a)–(e)). Growth
of hydroxylapatite has not been observed after 24 h.
DTA spectra of the dried gels indicate the glass transition
temperatures of samples as 611, 641, 691, 751 and 82 1C for
MZB-0. MZB-1, MBZ-2, MBZ-3 and MBZ-4 samples. In
order to confirm the observations of glass transition tempera-
ture from DTA data, XRD spectra of the dried gels have been
undertaken at 50 1C which confirms the amorphous nature.
Broad humps of crystallization temperatures have been
observed at 3961, 3601, 3421, 3331 and 317 1C for MZB-0.
MZB-1, MBZ-2, MBZ-3 and MBZ-4 samples. Broad humps
may be due to the formation of multiple crystalline phases.
3.1.2. FTIR studies
Some characteristic peaks have been observed at 470 cmÀ1
,
near about 710 cmÀ1
and 1080 cmÀ1
which can be assigned
to Si–O–Si bending, Si–O–Si bond and Si–O–Si asymmetrical
stretching. Vibration of P¼O has been observed near
1220 cmÀ1
along with C–O bond at 1420–1470 cmÀ1
. Some
BO3 vibrations at 1380 cmÀ1
indicate the presence of boron in
the samples. Peaks at 960-975 cmÀ1
show the presence of B–
O–M (M may be any metal). Two small humps at 876 cmÀ1
and 920 cmÀ1
are attributed to the vibrations of SiO4
4À
and
Si2O7
4À
bonds.. Humps at 619 cmÀ1
and near 986-1000 cm À1
represent the Mg band with influence of γ4PO4 vibrations.
When the samples are immersed into SBF and analyzed after
7 days and 14 days, some distinctive changes thus observed
are provided below;
Two sharp peaks at 601 and 558 cmÀ1
indicate the
presence of apatite (P–O bonds) on the surface of sample.
These peaks have become sharper when analyzed after 14
days. These peaks are the fingerprint peaks for HAp. These
results compliment the analysis of the XRD spectra.
Increase in magnitude of humps at 470 cmÀ1
, 801 and
1080 cmÀ1
(Si–O–Si) with time indicates the re-
polymerization of Si–O–Si layer and hence, confirms the
analysis of XRD studies.
3.1.3. FESEM and EDX studies
Powder of the sample has been filtered from SBF and
washed with acetone and DI water four times. In order to
remove the moisture, samples have been dried up to 60 1C.
Platinum coating has been undertaken to make the samples
conductive. Fig. 3 shows the difference in the morphology of
the representative samples before and after 14 days during in
vitro analysis. Change in the morphology can be attributed to
the formation of HAp layer on the surface of samples. FESEM
and EDX studies of sample have been undertaken after
confirmation of presence of apatite layer on the surface of
the samples from XRD and FTIR studies. FESEM images have
been taken after 14 days of in vitro analysis when apatite layer
is supposed to be fully grown on the surface of the samples.
FESEM micrographs confirm the analysis of XRD and FTIR
data. EDX results can provide the information about the
content of calcium and phosphorus in the samples. Ca/P ratio
from EDX analysis has been provided for representative
samples in Fig. 3. After 14 days, it has been observed that
Ca/P ratio of samples is in the range of 1.62–1.66. Ca/P ratio
of human bone is 1.66. In the light of this situation, it can be
concluded that EDX results also confirms the growth of HAp
on the surface of samples.
EDX study has been undertaken for all the samples. Atomic
percentage of constituent elements has been calculated from
nominal composition and compared with atomic percentage of
same elements as observed from EDX data (Table 2). Trends
of the atomic percentage of elements in nominal and experi-
mental compositions of the samples have been observed to be
similar. Moreover, the experimental and nominal values have
been observed to be close which suggest that the prepared
compositions are similar to the nominal compositions.
3.2. Concentration of ions and degradation behavior of the
system
Concentration of ions exchanged between the SBF and
sample interface has been investigated with the help of AAS
technique. Degradation of samples have been studied in citric
buffer and phosphate buffer saline (PBS) buffer solutions by
employing weight change, pH, XRD and AAS techniques.
Both are important parameters to understand the bioactive
behavior and also, to evaluate the samples for clinical
applications.
3.2.1. Concentration of ions
Exchange of ions between SBF solution and sample inter-
face is responsible for the formation of new phases and
variation in the morphology of the samples. AAS of decant
SBF has been investigated with 240FS Agilent Atomic
Absorption Spectrometer. Decant of SBF has been filtered
with .22 μm syringe filter. Dilution of SBF was run against the
standard solutions. Concentration of ions has been checked
during in vitro analysis and results have been presented in Fig.
4. With the increase in the content of borate and magnesium in
samples, following changes have been observed;
V. Anand et al. / Ceramics International 42 (2016) 3638–36513642

6. During in vitro analysis, ion concentration of silicon,
magnesium, zinc and boron ions in the solution have been
observed to increase within 50–60 h. It became almost
stable up to 250 h with marginal increase in concentration.
This slow dissolution of ions after 50–60 h may be due to
the different roles of zinc and magnesium and BO3 units in
the network.
The ion concentration of phosphorus and calcium has been
observed to be decreased in the decant within 24 h. during
in vitro analysis. But sudden increase in the concentration
of calcium ions has been observed after 50 h (Fig.4(c))
during in vitro analysis. It may be due to the dissociation of
CaCO3 into Caþ
and HCO3
À
ions(reaction provided in Eq.
(1)). After that, Ca and P ions concentration has been
observed to be regularly decreased which may be related to
the formation of apatite layer on the surface of samples.
It has been observed that with the increase in the concen-
tration of boron and magnesium in the samples, dissolution
rate decreases. It may be correlated with the decrease in
pore size of high content borate sample. In order to justify
Fig. 3. Representative FESEM micrographs and EDX results (a) Before in vitro and (b) after in vitro.
Table 2
Comparison of atomic percentage of elements in nominal composition with atomic percentage of elements as observed from EDX data.
Element MZB-0 (at%) MZB-1 (at%) MZB-2 (at%) MZB-3 (at%) MZB-4 (at%)
Nominal
composition
EDX Nominal
composition
EDX Nominal
composition
EDX Nominal
composition
EDX Nominal
composition
EDX
O 55.99 56.12 56.35 56.71 56.71 57.33 57.07 57.89 57.42 58.54
Na 14.60 15.78 13.17 14.01 11.74 13.01 10.30 11.31 8.87 9.07
Mg 0.72 0.77 1.43 1.62 2.15 2.31 2.86 2.94 3.58 3.89
Si 16.49 14.87 16.14 14.61 15.78 13.61 15.42 13.41 15.06 13.25
P 1.86 1.78 1.86 1.73 1.86 1.77 1.86 1.76 1.86 1.76
Ca 9.62 9.89 9.62 9.86 9.62 9.80 9.62 9.81 9.62 9.89
Zn 0.72 0.79 0.72 0.76 0.72 0.77 0.72 0.78 0.72 0.79
B 0.00 0.00 0.72 0.70 1.43 1.40 2.15 2.10 2.86 2.81
V. Anand et al. / Ceramics International 42 (2016) 3638–3651 3643

7. this statement regarding slower dissolution rate for samples
having high content of boron and magnesium, BET studies
have been performed. Details of the experimental set up
used for BET analysis has been provided by authors
elsewhere [2]. Evaluated pore size from BET study of
prepared samples is provided in Table 1. It can be inferred
from the table that prepared samples are porous in nature.
Without addition of boron, sample (MZB-0) has the highest
pore size. With the addition of the boron and magnesium,
pore size has been observed to decrease gradually (42 to
Fig. 4. Atomic Absorption Spectroscopy result of solution that soaks bioactive samples. Error bar indicates the standard deviation observed for three measurements.
V. Anand et al. / Ceramics International 42 (2016) 3638–36513644

8. 19 nm). These results indicate that slower dissolution rates
at higher contents of boron and magnesium in the samples
may be related to smaller pore size of the samples.
3.2.2. Degradation study
Degradation behavior of sample is an important parameter
which gives an idea about how fast sample will degrade in the
body. Degradation test of sample has been performed in two
different pH media; (i) PBS with pH 7.4 and (ii) Citric buffer
with pH 3.0. PBS has been selected because it is the most
common pH in the human body and citric buffer has been
selected because it is released by osteoblast cells during worst
conditions in the body [32]. Tests have been performed
without replacement of buffer solution after 120 h.
Weight change, pH, XRD and ion concentration of samples
under PBS and Citric buffers are the parameters investigated
under degradation study. When sample come in the contact
with buffer solution there is exchange of ions in between
sample and solution which disturb the stoichiometric weight
distribution of sample. This leads to the change in the net
weight of sample. If there is loss of ions from sample (leaching
of ions) then this may decrease the weight and loss of
crystalline phases and vice versa. With the leaching of ions,
pH of sample will also increase or decrease depending upon
the nature and concentration of ion leached into the solution.
XRD study may provide the information about the new phases
on the sample and AAS study can be used to check the nature
(anion or cation) and concentration of leached ions. Therefore,
weight loss, pH, XRD and AAS studies can be utilized as
Fig. 5. (a) Weight loss trend,(b) pH graph, (c) XRD spectra of samples treated with citric buffer, (d) XRD spectra of sample treated with PBS buffer show the
formation of ∎ Sodium Calcium Silicate and □ Silicon Phosphate, and (e) Concentration of different elements in buffer solutions. Error bars for (a) and (b) indicates
the standard deviation observed for three measurements.
V. Anand et al. / Ceramics International 42 (2016) 3638–3651 3645

9. complimentary techniques and they are very useful to find out
the exact degradation behavior of sample.
Weight loss percentage of sample has been calculated with
the formula given below;
WL% ¼
ðW1 ÀW2Þ
W1
 100 ð2Þ
where WL, W1 and W2 are weight loss, initial and final
weight of the sample. Graphical variation of weight loss data is
given in Fig. 5(a). It has been observed that MZB-0 has the
highest degradation rate with loss of 0.63 wt% whereas MZB-
4 has the lowest degradation rate with loss of 0.40 wt% in
citric buffer. Slow leaching may also be due to higher content
of boron and magnesium in the samples which may lead to
increase in the hydrophobic bridging oxygen atoms. The
MgO4
2À
units require some modifier cations in the matrix for
the purpose of charge balancing. Presence of borate units
increase the hydrophilic bridging oxygens. This may lead to
increase in the network connectivity which may further lead to
formation of compact structure with controlled degradation
rate. Weight loss in the PBS varies from À0.25(MZB-4) up to
-0.43(MZB-0) weight percent. This may be due to slow ion
leaching rate of MZB-4 because of its small pore size.
Negative weight loss has been reported in the case of PBS
buffer which indicates the gain of weight due to formation of
new crystalline phases (confirmed in XRD spectra Fig. 5(d)). It
has been observed by Merolli et.al that these newly formed
crystalline phases of sodium calcium silicate (JCPDF-78-1686)
and silicon phosphate (JCPDF-22-1320) are biocompatible and
bioactive [33]. XRD spectra of samples immersed in citric
buffer solution have shown amorphous nature of sample after
120 h. This may be due to high leaching of ions in the acidic
medium (pH¼3,citric buffer). It has been observed that pH of
citric buffer solution has increased from 3.00 up to 6.91 for
MZB-0 (maximum) and 5.32 for MZB-4 (minimum) as shown
in Fig. 5(b). This change in the pH value helps to initiate the
hydrolysis process of the samples. But in the case of PBS,
there is slight increase in the pH of solution from 7.91 (for
MZB-0) up to 8.66 (for MZB-4). Change in the pH may be
due to release of Ca and Mg ions from the whitlockite and
calcite phases. As discussed earlier, whitlockite is a stable
phase in the acidic pH (4.5–5),therefore, this phase start to
dissolve in citric buffer and PBS solution. Concentration of
leached ions in buffer solutions has been investigated with
AAS technique for solvent with the filtration from.22 μm filter
paper. Ionic release is an important factor which determine the
apatite growth and degradation of bioactive glass or ceramics.
Ceramics have different crystalline phases and are supposed to
take more time for the dissolution as compared to amorphous
glass system. It has been seen from AAS study that concentra-
tion of Si, P, Zn, Mg and B ions is higher in the case of citric
buffer solution as compared to PBS buffer(shown in Fig. 5(e)).
This observation supports higher change in pH value and
conversion of crystalline phases into amorphous in citric
buffer. It has been observed that silicon release is in the range
of 109.3–95.3 mg/l (Fig.5(e)) for citric buffer and 30.3–
23.5 mg/l for PBS buffer. Release of ions from sample is
beneficial to initiate different biological processes like osteo-
blast, cell growth and angiogenesis etc. For example, it has
been reported [32] that when ion concentration of silica ions is
between 0.1 and 100 mg/l, it is beneficial for stimulate the
osteoblast process. Similarly, calcium in the range of 13.1–
90 mg/l helps to initiate the osteoblast proliferation. In this
study, silicon and calcium ion concentration lies between 23.5
to 30.3 mg/l and 17.5 to 24.5 mg/l respectively. Magnesium is
also present in the extracelluer fluid in the concentration of 17–
25.5 mg/l and if its level is increased beyond 25 mg/l, it may
lead to muscular paralysis. In our study magnesium ion
concentration has been observed in between 11 and 20 mg/l
which is the comfortable zone for human tissues.
3.3. Swelling test
Swelling test is an important study to investigate the
behavior of pore size of sample when it comes in contact
with human plasma or PBS buffer. The swelling test of
prepared samples have been performed with conventional
gravimetric procedure as described below. Samples of known
weight are kept in 20 ml of PBS at 37 1C (pH¼7.4). Swollen
porous samples were drawn at various time intervals, dried
superficially by gentle contact with a filter paper and weighed
for the determination of wet weight as a function of the
immersion time. The swelling ratio percentage was calculated
as
%δw ¼
ðWa À WbÞ
Wb
 100 ð3Þ
Where wa and wb are the sample weights after and before
swelling, respectively. Each test was repeated three times for
each composition and results were expressed as average value
plus standard deviation (Fig. 6).
It is speculated that increase in swelling ratio and pore size
are correlated. Higher value of swelling ratio percentage and
pore size of samples for low content borate samples support
Fig. 6. Swelling ratio percentage of prepared bioactive samples. Error bar
indicates the standard deviation observed for three measurements.
V. Anand et al. / Ceramics International 42 (2016) 3638–36513646

10. this inference (Fig. 6 and Table 1). Capillaries in the pores
avail nutrients from culture media more effectively. Swelling
can enhance the cell adhesion.
3.4. Drug release
Drug release property can be used to explore the possibi-
lities of samples as drug carrier agents. Gentamycin as an
antibiotic has been tested for drug release study of the samples
because this drug has good activity against gram negative
microorganisms. 1 gm of prepared sample has been immersed
in 20 ml of gentamycin solution. After gentamicin was
incorporated into sample, sample has been kept in the solution
up to 24 h. After filtering the powder and drying at 40 1C up to
24 h, release of gentamycin from the drug-loaded bioactive
sample has been investigated in incubator at 37 1C. One gram
of powder has been dipped in the 20 ml of SBF under 37 1C.
Gentamycin release was determined by UV analysis. The
release medium was withdrawn at the predetermined time
intervals and replaced with same amount of fresh SBF solution
each time. During the drug release mechanism, all the prepared
samples show quick release in first hour and then there is
decrease in the rate of release of drug in SBF (shown in Fig.
7). All the samples show similar drug release behavior as
reported by mesoporus channel [34]. Reported BET data
(Table 1) indicate the mesoporus nature of our samples. This
study shows that prepared samples have good response in drug
delivery phenomena and it is due to their mesoporus behavior.
3.5. Antimicrobial activity
Tendency to kill the microorganisms has been studied
against six different gram positive and gram negative micro-
organisms.Multiple drug-resistant microorganisms such as
MRSA have increased in the world [35]. The development
of new antimicrobials is the emerging challenge to answer the
problem posed by resistant microorganisms. Keeping the
resistance factor in mind and the demand for new antimicrobial
agents, we have tested our samples for antimicrobial potential.
Antimicrobial results are provided in Fig. 8.
3.5.1. Inoculum preparation
A loopful of isolated colonies was inoculated into 5 ml
nutrient broth and incubated at 37 1C for 4 h. The turbidity of
actively growing microbial suspension has been adjusted to
match the turbidity standard of 0.5 Mc Farland units prepared
by mixing 0.5 ml of 1.75% (w/v) barium chloride dihydrate
(BaCl2 Á 2H2O) to 99.5 ml of 0.18 M (v/v) sulfuric acid during
constant stirring
3.5.2. Test organisms
The reference strains of bacteria: Staphylococcus aureus
(MTCC-740) Klebsiella pneumonia sub sp. pneumoniae
(MTCC-109), Pseudomonas aeruginosa (MTCC-741),)
Fig. 7. Drug release study of samples. Error bar indicates the standard
deviation observed for three measurements.
Fig. 8. (a) Representative figure for microbial activity and (b) bar graphs of
different microbial activity with samples. Error bar indicates the standard
deviation observed for three measurements.
V. Anand et al. / Ceramics International 42 (2016) 3638–3651 3647

11. Salmonella typhimurium (MTCC-1251) and yeast strain:
Candida albicans (MTCC-227) have been obtained from
Microbial Type Culture Collection (MTCC), Institute of
Microbial Technology (IMTECH), Chandigarh, India and the
clinical isolate MRSA has been obtained from Post graduate
Institute of Medical Education and Research, (PGIMER),
Chandigarh, India.
3.6. Antimicrobial activity by agar well diffusion assay
The plates containing Muller Hinton agar medium have
been spread with 0.1 ml of the microbial inoculum. Wells
(8 mm diameter) have been cut from agar plates using
sterilized stainless steel cork borer and filled with 0.1 ml of
the fungal extract. The plates have been incubated at 37 1C for
24 h and diameter of resultant zone of each combination of
extract and bacterial strains inhibition has been measured [36].
Experiments have been run in triplicate for each combination
of extract and bacterial strains. There are several resources that
can be tapped for useful products such as antibiotics. The
bacterial cultures used in the present study are responsible for
causing gastrointestinal tract and respiratory infections.
All the samples have been found to be active against almost
all the microorganisms tested. S.aureus was found to be the
most sensitive organism and the inhibition zone was found to
be in the range of 21–25 mm for boron containing samples.
Gram negative bacteria acquire resistance more readily due to
their outer membrane which contains narrow porin channels
which retard the entry into the cell, of even small hydrophilic
compounds, a lipopolysaccaride moiety which slows down the
trans membrane diffusion of lipopolyphilic antibiotics and they
often possess a multidrug efflux pump which eliminates many
antibiotics from the cells causing several diseases. All the
boron containing samples have shown good antimicrobial
potential against gram negative bacteria viz K.pneumoniae,
P.aeruginosa and S.typhimurium with zone of inhibition
ranging from 16 to 20 mm, 12 to 20 mm and 04 to 14 mm
respectively. The importance of the reported study became
paramount when resistant strains like MRSA were also found
sensitive to the prepared samples which were not reported
earlier. Prepared boron containing samples have shown not
only activity against bacterial cultures but have also shown
good activity against yeast C.albicans with zone of inhibition
ranging from 15 to 21 mm. Results have shown that our
samples can be potent antimicrobial agents which can be
further exploited for various pharmaceutical processes.
3.7. Cell cytotoxicity and culture studies
Toxicity and cell attachment (through cell culture investi-
gations) have been studied to investigate the friendly beha-
vior of sample with cells. Samples have been observed to be
non-cytotoxic as per the procedure reported by authors
elsewhere [37]. MTT (3-[(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyl] tetrazolium bromide) assay has been used for
this study. 10 mL sheep blood has been taken into injection
syringe containing 3 mL Alsever's solution (anticoagulant)
which was subsequently transferred to sterile centrifuge
tubes. The blood has been centrifuged at 1600g at room
temperature for 20 min to separate the plasma from the cells.
The supernatant has been discarded and 6 mL PBS was added
which was further centrifuged. The red blood cells (RBCs)
have been washed thrice with PBS by centrifugation techni-
que and the pellet has been re-suspended in 6 mL of PBS.
Various dilutions of these cells using PBS have been prepared
and counted with the help of a haemocytometer under optical
microscope so as to obtain cells equivalent to
1 Â 105
CFU/mL. The following formula has been used to
determine the required number of cells;
Number of cells=mL ¼ Number of cells counted in 25 squares
 Dilution factor  104
ð1Þ
The cell suspension thus prepared has been dispensed into
Elisa plates (100 mL/well) and incubated at 37 1C for over-
night. The supernatant has been removed carefully and 200 mL
of the compound (sample dissolved in DMSO) has been added
and incubated further for 24 h. Supernatant has been removed
again and added to 20 mL MTT solutions (5 mg/mL) to each
well and incubated further for 3 h at 37 1C on orbital shaker at
60 rpm. After incubation, the supernatant has been removed
without disturbing the cells and 50 mL DMSO has been added
to each well to dissolve the. The wells with untreated cells
have served as control. In the presented study,viable cell
percentage of samples has been calculated by absorbance
intensity. For MZB-0, MZB-1, MZB-2, MZB-3 and MZB-4,
the observed cell viabilities are 70.3%, 70.3%, 79.3%, 79.3%,
79.3% and 79.3% respectively (Fig. 9). The obtained results
suggest that all the prepared samples are non-toxic in nature. It
can also be inferred from the results that increase in the content
of borate and magnesium reduce the toxicity of sample.
Only HAp formation ability of material is not enough to
make it an implant material. Before implantation it is also
important to check how it reacts with human osteoblast cells.
In order to check the behavior of samples with living cell, the
human osteosarcoma cell line has been obtained from National
Center of Cell Science, Pune,India. DMEM (Dulbecco's
Modified Eagle's Medium) has been used with FBS(fetal
bovine serum)10%, streptomycin and gentamycin 100 U mlÀ1
each to maintain cell lines under 37 1C incubation with humid
environment containing 5% CO2. MTT assay has been used to
Fig. 9. Cell viability of sample during cytotoxicity test. Error bar indicates the
standard deviation observed for three measurements.
V. Anand et al. / Ceramics International 42 (2016) 3638–36513648

12. check the cell integrity. 24 well plates have been used to bind
the MG 63 cell with samples. MG63 cells are seeded on the
sterilized plate with concentration 2 Â 104
cell mlÀ1
. Samples
are kept under 37 1C with 5% CO2 environment for 96 h.
Tissue culture treated plastic cover slip (Theromanox) has been
used to grow controlled culture. Glass slices have been kept in
triplicate in 24 well plates. Each well plate has been filled with
500 μl volume of lymphocyte suspension at the rate of
2 Â 104
cell mlÀ1
. Plates have been incubated for 96 h.
500 μl MTT (2 mg mlÀ1
) has been added to the plates before
4 h. for termination. After 4 h, blue colored formazan had
appeared in each well plates which was studied with 570 nm
UV radiations with the help of Labsystem Multiskan EX
ELISA reader against a reagent.
Fig. 10 shows the cell viability of samples with respect to
MG63 cell. Samples have been compared with commercial
available culture plate. It has been observed that all the
prepared samples successfully provide the positive environ-
ment for cell growth (high absorbance indicate good cell
growth). Leaching of ions have shown impact on the biological
behavior of samples. Cell proliferation remains good if
leaching of ions is smooth and regular. Sudden increase in
the concentration of ions may cause the death of cell. Cell
viability of all the prepared samples is good and hence, this
observation compliments the results reported in Fig. 4. It has
been already established that presence of zinc ions play
important role in the growth of cell. It can be seen in Fig.
10 that boron and magnesium containing samples enhance the
cell proliferation. Observed trends in Fig. 10 may be due to the
smooth dissolution of boron and zinc ions as discussed under
the section concentration of ions and degradation behavior of
the system.
Many authors [38–42] have investigated the growth of
hydroxylapatite layer on silica based bioactive samples. It
has been observed that many samples took more than 14 days
to initiate the growth of hydroxylapatite layer. In the presented
work, authors have reported the faster growth of hydroxyla-
patite layer (7th day) during in vitro analysis. Faster is the
growth of hydroxylapatite on the surface of sample, quicker
will be the bond formation between host and implant material.
This will lead to recovery of damaged bone in shorter span of
time. Kapoor et.al. and Kansal et al. [43,44] have studied the
degradation behavior of amorphous bioactive samples in two
different pH buffer solutions. pH values used were 3 and
7.4 which are the same values as used by the authors in the
presented study. Ion leaching for Si, Ca and P observed by
Kapoor et.al. and Kansal et.al was higher as compared to the
ion leaching observed by authors for the same ions. Controlled
leaching of ions is an important property to improve the
therapeutic efficiency of the treatment. Controlled leaching of
ions was observed for the presented system which may be
attributed to crystalline nature of the samples. Very few
authors [45–47] have reported the cell viability greater than
control sample. Authors have observed 35% higher viability as
compared to control for MZB-4 sample. It indicates that
prepared sample helps in the proliferation of MG 63 cell lines.
Growth of MG 63 cells initiate the osteoblast (bone formation)
process. More will be the growth of cells, faster will be the
repair of the damaged bone. These results indicate the
significant contribution by the authors in the presented work
in terms of growth of hydroxylapatite layer, degradation
behavior and cell viability properties of the bioactive materials.
4. Conclusions
The prepared samples have shown good bioactivity beha-
vior. This feature has been confirmed by the presence of
apatite peaks in XRD and presence of P–O bonds in FTIR
spectra at 558 and 601 cmÀ1
during in vitro studies. Further-
more, FESEM and AAS studies compliments the analysis of
XRD and FTIR spectra. During the immersion of samples in
SBF, calcium to phosphorus ratio (from EDX data) indicate the
growth of the apatite phase. It has been inferred from BET
studies that growth of apatite depends upon the porous nature
of the samples, High porosity increase the contact area of
sample with SBF which results in the increase in the apatite
Fig. 10. (a) Cell viability bar graph. Error bar indicates the standard deviation observed for three measurements and (b) Representative optical image (at 40 Â
magnification) of MG63 cell line grown on the surface of sample.
V. Anand et al. / Ceramics International 42 (2016) 3638–3651 3649

13. growth. Pore size has been found to be lowest in the sample
containing high content of borate. All the prepared samples
have been observed to be non-toxic in nature with more than
70.3% viable cells. Attachment with MG63 cell line shows that
samples provide the positive environment for the growth of
cell line. It has been observed that increase in the content of
borate and magnesium leads to enhanced percentage of viable
cells. Antimicrobial activity indicate the resistive nature of
samples towards microorganisms.MZB-4 sample can be con-
sidered as the best sample prepared in the laboratory due to the
following reasons. This sample has good bioactivity, slow
degradation, $87% drug release, 79.3% cell viability, excel-
lent cell proliferation and good tendency to kill microorgan-
isms. Results indicate that samples prepared in this study can
have potential clinical applications as osteoconductive carriers
for treating bone infection. Authors recommend the MZB-4
composition for further in vivo testing for clinical applications.
Acknowledgments
The authors Vikas Anand and Kulwinder Kaur are grateful
to the financial assistance provided by the UGC, New Delhi
(India) through SRF (NET)[F.17-74/2008(SA-I)] and DST,
New Delhi (India) through INSPIRE program SRF[IF-120620]
respectively.
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