Ceramic International 2016

Magnesium and silver doped CaO–Na2O–SiO2–P2O5 bioceramic
nanoparticles as implant materials
Kulwinder Kaur a
, K.J. Singh a,n
, Vikas Anand a
, Gaurav Bhatia b
, Sukhdev Singh b
,
Harpreet Kaur c
, Daljit Singh Arora c
a
Department of Physics, Guru Nanak Dev University, Amritsar 143005, India
b
Department of Molecular Biology & Biochemistry, Guru Nanak Dev University, Amritsar 143005, India
c
Department of Microbiology, Guru Nanak Dev University, Amritsar 143005, India
a r t i c l e i n f o
Article history:
Received 9 March 2016
Received in revised form
28 April 2016
Accepted 1 May 2016
Available online 2 May 2016
Keywords:
Sol–gel processes
MgO
Biomedical applications
Hydroxyl apatite
a b s t r a c t
Bioceramic nanoparticles of composition xMgO-yAg2O-(40Àx)CaO-(20.5Ày)Na2O-35.5SiO2-4P2O5 ( x
and y values are 0, 2, 4, 6, 8 and 10 mol%) have been prepared in the laboratory by using quick alkali-
mediated sol gel method. Role of silver along with magnesium on the the properties of above mentioned
bioceramics has been investigated by using X-Ray Diffraction, Transmission Electron Microscopy, Fourier
Transform Infrared, Brunauer-Emmett-Teller and Atomic Absorption Spectroscopy techniques. De-
gradation behavior in Citric Buffer and Simulated Body Fluid has also been investigated. Drug delivery
property of prepared samples has been checked with UV-Visible along with Brunauer-Emmett-Teller
techniques for antibiotic drug gentamycin. Antimicrobial response against six different microorganisms
including Methicillin-Resistant Staphylococcus aureus has been checked. Controlled release of silver ions
from our samples to demonstrate their ability to treat infection has been evaluated with the help of
heterogeneous model of diffusion controlled mechanism. Cytotoxicity and cell culture (with MG 63 cell
lines) studies have also been conducted. It has been observed that samples provide friendly environment
for the growth of MG 63 cell lines. Experiments have been conducted to find the suitable chemical
composition of implant material with excellent bone regeneration and antimicrobial properties.
& 2016 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
1. Introduction
Among various types of biomaterials, bioceramics have been
found to be beneficial for repair of damaged and diseased bone
tissues due to their high biocompatibility [1–3]. They form hy-
droxyl apatite (HAp) layer on their surface by release of silica via
interfacial reaction with simulated body fluid (SBF) [4,5]. This layer
can be observed both in vitro and in vivo experiments. If the rate
of formation of HAp layer is faster, repair of damaged tissue will be
quicker. This is the desired property of the implant material for
clinical applications. This property is related directly to the surface
area and porosity of the bioceramics. Therefore, recent studies
have been oriented towards the production of porous bioceramics
in nanoscale range having high surface area to volume ratio in
order to enhance bioactivity. HAp is chemically similar to in-
organic phase of human bone which makes it an established
material to replace human bone. Due to good osteoconductive,
osteoproductive and osteoinductive nature, bioceramics can be
used in different biomedical applications including bone graft
extenders and bone defect filling materials. Properties of bioma-
terials can be tailored easily by varying the composition [6]. Efforts
have been undertaken by many researchers to prepare bio-
ceramics with improved properties in several areas including anti-
inflammatory, antibacterial and bone growth rate. These can be
achieved by incorporating several elements such as zinc, magne-
sium, strontium, silver and copper in the composition of implant
material [7–13]. Moreover, bioceramics should have thermal and
chemical stability and good degradation rate [14].
Degradation is an important parameter to control the reactivity
of bioceramics in the medium (SBF). High degradation rate leads to
high release rate of ions from the surface of the sample to the
solution. This may kill the cells surrounding implant material and
also decrease the mechanical strength of the implant. On the other
hand, very low degradation rate may slow down the release of
ions which may further lead to slow growth of HAp layer on the
surface of sample. Therefore, bioceramics must have optimum
degradation rate.
Mg is widely used in the composition of the bioceramics to
enhance the bone regeneration and control the degradation rate
[15–17]. Mg has stimulatory effects on the bone development and
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ceramint
Ceramics International
http://dx.doi.org/10.1016/j.ceramint.2016.05.001
0272-8842/& 2016 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
n
Corresponding author.
E-mail address: kanwarjitsingh@yahoo.com (K.J. Singh).
Ceramics International 42 (2016) 12651–12662

maintenance of polymerized silica network. Mg as well as Mg ions
naturally exists in the human body and participate in the structure
of the body through their presence in the bone. Mg is used by the
body for many functions including (i) glucose and fat breakdown
(ii) production of proteins, enzymes and antioxidants (iii) creation,
stabilization and repair of DNA and RNA (iv)regulation of choles-
terol production (v) ions of Mg as enzyme co-factors to regulate
several biochemical reactions [18]. Deficiency of Mg in the body
slows down synthesis of DNA, osteoporosis and metabolic syn-
drome. Many researchers have investigated the effect of Mg as
trace element for improving the degradation rate [19]. Bacteria
caused diseases have attracted wide attention. Bioceramics do not
have ability to protect against microorganisms (fungi etc.) [20].
Therefore during implantation of bioceramics in human body, in-
fection, allergic reactions, microbial flora depletion, prophylaxis
and bacterial resistance are most commonly encountered pro-
blems. These problems can cause failure of the implant. Bio-
ceramics having excellent antibacterial and antifungal properties
through incorporation of antibacterial agents/ions (Agþ
, Cu2 þ
etc.)
are always the preferred candidates as implant materials [8,21].
During dissolution of bioceramics, these ions will be released and
they can induce additional therapeutic effects [10,15]. Ag is most
commonly used antibacterial agent in the form of Agþ
exhibiting
low toxicity towards cells and tissues [22–25]. Charge to size ratio
of silver is similar to sodium that results in a similar release be-
havior for antibacterial action. Ag doped bioceramics have bac-
teriostatic potential and they can induce bacteria reactions. Several
researchers have undertaken studies of magnesium and silver as
individual elements separately in various materials as anti-
bacterial, bone development and maintenance agents [2,6,17,26–
31]. These observations suggest that both Mg and Ag are very
important elements for composition of implant material for bone
regeneration applications and a successful implant material may
require the presence of both of the elements. In the light of these
observations, authors have chosen to study the effect of simulta-
neous presence of Mg and Ag in the investigated bioactive system.
In the presented work, we report an easy way to prepare bio-
ceramics containing Mg and Ag (both) by quick alkali -mediated
sol-gel method [9,32]. Quick alkali mediated sol gel method has
been chosen over conventional sol-gel method to achieve fast
gelation ($1 min). Bone regeneration ability of the prepared
samples has been checked after 1 day of incubation in SBF. Anti-
bacterial properties of the samples have been investigated in de-
tail. For this purpose, six different gram positive and gram nega-
tive microorganisms have been selected. To check the diffusion
controlled release of therapeutic ion (silver), heterogeneous model
has been fitted into the Atomic Absorption Spectroscopy (AAS)
data. In vitro cell attachment of bioceramics as a drug delivery
vehicle, antibacterial property, controlled degradation rate and
good cell viability can be very useful studies for estimating the
practical utility of the prepared samples as successful implant
materials in human body. Authors have reported the data con-
cerning the above mentioned studies.
2. Material and methods
2.1. Sample preparation
Tetraethyl orthosilicate(TEOS), triethyl phosphate(TEP), calcium
nitrate tetrahydrate, sodium nitrate, silver nitrate and magnesium
nitrate hexahydrate (Merck, AR grade,) have been used as source
materials for silicon dioxide, phosphorus penta oxide, calcium
oxide, sodium oxide, silver and magnesium oxide. Nitric acid,
ethanol and ammonium hydroxide solutions have been used as
hydrolysis and condensation agents.
Samples of the composition xMgO-yAg2O-(40Àx)CaO-(20.5À
y)Na2O-35.5SiO2-4P2O5 (x and y values are 0, 2, 4, 6, 8 and
10 mol%) have been prepared in the laboratory by quick alkali
mediated sol-gel process. MgO and Ag2O have been used to re-
place CaO and Na2O as shown in Table 1. Initially, TEOS has been
hydrolyzed in the mixture of 2 M nitric acid, water and ethanol for
1 h which creates excess ethanol. This has been followed by ad-
dition of TEP, calcium nitrate tetrahydrate, magnesium nitrate
hexahydrate, sodium nitrate and silver nitrate at the intervals of
30 min each. This has been followed by stirring for 30 min which
has been further followed by ultra-sonication by using Citizon
Digital Ultrasonic Cleaner (CD 4820). 1 M ammonium hydroxide
has been added drop wise to the solution with mechanical stirring
for quick gelling of precursor materials. Ammonium hydroxide
solution provide excess −OH ions, transform solution into gel with
in fraction of minutes which reduce the aggregation. A schematic
diagram of mechanism is given in Fig. 1. Gelation of the compo-
nents has occurred within 1 min after addition of ammonia solu-
tion. Prepared gel has been dried at 75 °C for 48 h. followed by
calcination at 700 °C for 3 h. XRD diffractograms of calcined
samples have shown the majority of Na-Ca-Si phase along with
crystallization phase of silver.
2.2. Characterization techniques
2.2.1. X-Ray diffraction
X-ray diffraction (XRD) diffractograms of prepared samples
have been obtained by using Bruker D8 focus X-ray Diffractometer,
Germany equipped with Cu Kα (λ¼1.54 nm). Target has been set
to power level of 40 mV and 30 mA in 2θ range of 10–70°. Pre-
sence of different phases has been identified by using JCPDS data
files in Pcpdfwin software.
2.2.2. Fourier transform infrared spectroscopy
Fourier transform infrared (FTIR) spectra of prepared samples
have been recorded by using Perkin Elmer C92035, Germany.
Absorption spectra have been obtained from 4000 to 450 cmÀ1
.
Baseline of all the spectra has been corrected by using ORIGIN
8.5 software.
2.2.3. Transmission electron microscopy
Transmission electron microscopy (TEM) study has been
Table 1
Chemical composition and surface properties of prepared samples.
Sample code MgO Ag2O CaO Na2O P2O5 SiO2 BET (drug loaded) BET (drug released)
Pore size (D) Surface area (SBET) (m2
/g) Pore size(D) Surface area (SBET) (m2
/g)
BMA-0 0 0 40 20.5 4 35.5 12 32 42 58
BMA-1 2 2 38 18.5 4 35.5 14 34 32 46
BMA-2 4 4 36 16.5 4 35.5 13 37 35 44
BMA-3 6 6 34 14.5 4 35.5 16 35 34 45
BMA-4 8 8 32 12.5 4 35.5 18 38 37 47
BMA-5 10 10 30 10.5 4 35.5 17 39 38 44
K. Kaur et al. / Ceramics International 42 (2016) 12651–1266212652

undertaken to investigate the morphological properties of the
prepared samples. Suspension of samples has been prepared in
ethanol with 99.9% purity level as a solvent. Prepared suspension
has been analyzed by JEOL JEM 1200 transmission electron
microscope.
2.2.4. Atomic absorption spectroscopy
Prepared samples have been immersed in SBF and citric buffer
solutions at 37 °C (0.1 g sample in 25 mL of SBF) for different time
intervals. Release of calcium, magnesium, sodium, silver, silicon
and phosphorus ions from the samples in SBF and citric buffer
have been investigated by using 240 FS, Agilent, Atomic Absorp-
tion Spectrophotometer. Aliquots of 10 mL have been filtered
through 0.22 mm syringe filter and they have been analyzed by
using AAS.
To check the amount of silver release from the surface of
samples in the SBF [33], the commonly used model is hetero-
geneous model [34,35]. In this model, leaching of ions from the
samples is governed by (i) diffusion controlled ion exchange (ii)
dissolution of the sample. These are the same first two stages
which are involved in bioactivity mechanism given by Hench [36].
In the first stage, alkali and alkaline earth ions of the sample are
replaced by Hþ
or H3Oþ
ions of the solution. This exchange pro-
cess varies inversely with time. This can be expressed in this
model by equation:
= ( )Q kt 1
1
2
where, Q¼Cumulative silver ion release concentration.
t¼Soaking time in h.
k¼Multiplying coefficient.
To find the value of k, Q is plotted against t
1
2 and a straight line
is fitted. Slope of the line gives the value of k. In addition, dis-
solution of ions is considered to follow the heterogeneous model if
correlation coefficient (R2
) is greater than 0.95 [33]. Therefore,
values of R2
have also been determined.
2.2.5. Brunauer-emmett-teller study
Brunauer-Emmett-Teller (BET) study of prepared samples for
pore size and surface area has been undertaken by micrometrics
ASAP 2020. Results for surface area using all adsorption data
points 0.01–1.0 (total points 46) are obtained by adsorption-des-
orption phenomena of N2. Pore size has been obtained by Barret-
Joyner-Halenda (BJH) desorption method (relative pressure 1.0–
0.12, total points 16).
2.2.6. UV–visible spectroscopy
For drug release property, UV-Visible data has been recorded at
room temperature on UV-3600 SHIMADZU UV–vis–NIR
spectrophotometer and for biological studies of the prepared
samples, Biorad 680-XR, Japan with 570 and 590 nm wavelengths
of UV–visible range has been used.
2.2.7. Field emission scanning electron microscopy
Field Emission Scanning Electron Microscopy (FESEM) study
has been carried out by ZEISS SUPERA 55. In order to get FESEM
images, samples have been washed with ethanol and DI water four
times. Moisture has been removed from samples by drying them
at 37 °C. Platinum coating has been used to make the samples
conductive.
3. Results and discussion
3.1. Estimation of bioactive nature of samples
The in vitro biological response of the samples for bone re-
generation properties has been investigated by using Kokubo's SBF
[33]. 0.1 g samples in 25 mL SBF solution have been sealed in
sterilized plastic containers and placed in an oven at 37 °C. Con-
tainers have been agitated thrice a day, to prevent agglomeration.
Analysis of samples has been undertaken at different time inter-
vals i. e. after 1, 5 and 12 days. Powders have been rinsed with
deionized water and then dried at 37 °C for 1 day. Dried samples
have been investigated by using XRD and FTIR techniques to check
the growth of bone mineral hydroxyl apatite layer. Experiments
have been performed in duplicate to ensure the accuracy of
results.
3.1.1. Before immersion in SBF
All the prepared samples have been observed to be crystalline
in nature with combeite as a major phase (JCPDS no.78–1650)
(fig. 2(a)–(f)). Sharp peaks at 38.12°, 44.27°, 64.48° and 77.41° in-
dicate the presence of silver (JCPDS no. 001-1167) (Fig. 2(b)–(f)).
Peaks at 29.29°, 38.40°, 44.39° and 64.49° show the presence of
magnesium oxide (JCPDS no. 030-0794) (Fig. 2(b)-(f)) whereas,
peaks at 26.8° and 23.9° indicate the presence of magnesium
phosphate (JCPDS no. 043-0225) (Fig. 2(b)-(d)) at the expense of
the phase calcium carbonate (JCPDS no. 03-0593) (Fig. 2(a)-(b)).
This result indicate the incorporation of magnesium in the matrix
at the place of calcium. Magnesium nitrate hexahydrate as pre-
cursor of magnesium generate Mg2þ
ions which interact with the
−
PO4
3
ions of TEP. Interaction of these two ions with each other
form magnesium phosphate as a distinct phase as per the fol-
lowing equation:
+ → ( ) ( )+ −
3Mg 2PO Mg PO 22
4
3
3 4 2
In some diffractograms, the peaks of magnesium and calcium
Fig. 1. Schematic diagram of mechanism.
K. Kaur et al. / Ceramics International 42 (2016) 12651–12662 12653

Fig. 2. XRD spectra of sample before and after in vitro analyses of samples ( Combeite, ■ Hydroxylapatite, ● Calcium Carbonate, ▲Silver, ▼ Magnesium phosphate, △
Magnesium oxide).

phases have been superimposed by high intensity silver peaks
(Fig. 2(d)-(f)).
TEM studies have been used to confirm the results of XRD.
Fig. 3(a) is a representative TEM micrograph for BMA-2 sample.
Fast Fourier Transform (FFT) calculated hkl values and High Re-
solution TEM (HRTEM) have been provided in the Fig. 3(b) and
(c) respectively. For the comparison, hkl values of corresponding
2θ° calculated from XRD is given in Table 3(d). It can be seen that
hkl values of (024), (211), (300), (042) and (220) during FFT cal-
culations are well matched with hkl calculated from XRD data for
combeite phase (Table 3(d)) which confirms the authenticity of
XRD results.
FTIR absorption spectra of all prepared samples have been
shown in Fig. 4. Observed peaks with their assigned structural
units are also provided in Table 2. It has been seen that the most
intense band for all prepared samples is in the region
850–1250 cmÀ1
(Fig. 4(a)-(f)) which may be assigned to different
stretching modes in between silicon and oxygen atoms (bridging
and non-bridging ) of SiO4 [37,38]. In case of BMA-0 (Fig. 4(a)),
BMA-2 (Fig. 4(c)) and BMA-3 (Fig. 4(d)) compositions, this band
splits into two bands at ∼936 cmÀ1
and ∼1037 cmÀ1
. Lower fre-
quency (∼936 cmÀ1
) band is due to Si-O asymmetric stretching
mode of the non-bridging oxygen atoms and higher frequency (∼
1037 cmÀ1
) band is due to Si-O asymmetric stretching mode of the
bridging oxygen atoms [39,40]. With increase in the content of
modifier oxides (Mg and Ag), splitting of bands have disappeared.
This may be due to the phosphate group present in the matrix
which increases the number of bridging oxygen atoms by using
modifier cations (silver and magnesium) to compensate the charge
of −
PO4
3
[16]. Presence of band at 877 cmÀ1
is due to P-O-H group
of Mg3 (PO4)2 and Ca3 (PO4)2 units [38]. This vibration indicate that
magnesium is fully incorporated in the matrix which further lead
to the contraction in the matrix due to replacement of lower ionic
radii of Mg2 þ
(0.78 Å) with Ca2 þ
(1.06 Å) [41].
3.1.2. After immersion in SBF
Bioactivity of prepared samples has been evaluated upto 12
days after immersion in SBF. SBF have ionic concentration
equivalent to human blood plasma. XRD diffractograms and FTIR
spectra show that HAp layer generation starts in 1 day, which is
the key confirmation of bioactivity.
XRD diffractograms confirms the formation of HAp layer (JCPDS
no. 072-1243) with sharp peaks at ∼25.8° and 31.6° and reduction
in combeite phase intensity after 1 day of immersion in SBF. For-
mation of HAp layer is due to exchange of ions between the
powdered sample and SBF. With the increase in the content of Mg
and Ag, HAp layer formation is slow. This may be due to magne-
sium ions ( )−
MgO4
2
entering in the silica matrix. −
MgO4
2
units in-
crease the network connectivity of the silica matrix by utilizing
modifier cations for charge balancing purposes. Therefore, cations
will no longer produce non-bridging oxygens (NBOs). NBOs in-
teract with ions of SBF due to their hydrophilic nature while
bridging oxygens are hydrophobic in nature which makes them
least reactive with ions of SBF [16]. Presence of NBOs enhance the
bioactivity of the samples by decreasing the network connectivity.
In case of BMA-0 and BMA-1 samples, modifier cations are not
dominating as in case of rest of the samples. Presence of calcium
carbonate phase (JCPDS no. 003-0593) is confirmed along with the
magnesium phosphate (JCPDS no. 043-0225) in BMA-1 sample.
BMA-0 has lowest network connectivity in terms of modifiers ions
present which causes the increase in the bioactivity of sample.
With the increase in the content of Mg, Ca2þ
ions are replaced by
Mg2 þ
ions which leads to the formation of magnesium phosphate
and magnesium oxide.
Bioactivity of the samples has also been confirmed by FTIR
absorption spectra (Fig. 4). Spectra show the formation of strong
−
PO4
3
band at ∼559 and∼601 cmÀ1
, which confirm the formation of
HAp layer [42]. Therefore, it can be concluded that FTIR spectra
compliments the analysis of XRD data which indicate the bioactive
nature of the samples.
3.2. Degradation studies
Degradation tests have been performed in citric (pH¼3.0) and
SBF (pH¼7.4) buffer solutions. Low pH citric buffer solution has
been selected for degradation studies because osteoclasts release
this acid under worst conditions in the human body. Experiments
have been undertaken at 37 °C without replacement of solutions.
Samples have been taken after a period of 120 h. for studies. Solid
and liquid phases have been separated by filtration and then wa-
shed with distilled water and dried in the oven. A relative weight
loss percentage of prepared samples in both solutions have been
calculated by using relation:
=
−
×
( )
W %
W W
W
100
3
L
1 2
1
where, WL ¼Weight loss of the sample.
W1 ¼Weight of the sample before immersion in buffer
solutions.
W2 ¼Weight of the each sample after 120 h after being dried.
Fig. 3. (a) Representative TEM image, (b) Diffraction pattern, (c) FFT micrographs, (d) XRD hkl table.

Fig. 4. FTIR spectra of sample before and after in vitro analysis.

Degradation study of the samples has been undertaken with
the help of the following parameters; (1) Weight loss, (2) pH
change, (3) Phase variation and (4) Change in ion concentration.
These four parameters are interconnected to each other. Change in
weight is due to the exchange of ions in between surface of sample
and solution. This ion exchange leads to change in pH value of
solution which further causes formation of new stable phases.
Higher weight loss leads to rapid change in pH with the increase in
the concentration of ions in the solution.
In the SBF and citric buffer sample, BMA-5 reported highest
weight loss with change in pH upto 8.21(in SBF) and 4.91(in citric
buffer) owing to highest ion release rate. XRD analysis of degraded
samples in citric buffer and SBF has been undertaken and it is
found that all phases present earlier in the samples start to de-
grade rapidly in citric buffer (Fig. 5(e)) as compared to SBF (Fig. 2
(a)–(f) after 5 days). It can be observed from the Fig. 5(e) BMA-5
sample became amorphous except presence of silver peaks.
Combeite phase dissolves by release of Naþ
and formed a new
phase calcium silicate (JCPDS no. 03-0777) in sample BMA-0. XRD
patterns (Fig. 5(e)) has confirmed the amorphous nature which is
due to high degradation rate. Long time existence of silver peaks is
good for last long antimicrobial effect.
In the case of SBF (pH¼7.40), silica matrix breakdown and the
ions like Naþ
and −
SiO4 are released from the surface of the sample
to the solution rapidly. Other ions like Ca2 þ
and −
PO4
3
easily mi-
grate from the surface of the sample during contact of sample with
SBF and increase the concentration of Ca2 þ
and −
PO4
3
ions which
form the precipitate of HAp layer on the surface of SiO2 rich layer
[47,48]. Formation of HAp layer is similar as confirmed by XRD
diffractograms in Fig. 2. Modifier ions from the sample through
this silica rich layer are continuously released and they can per-
form therapeutic action. Brauer [16] reported that modifier ions
are linked with NBOs. Therefore, they can be released rapidly
during the first few minutes to hrs. Change in the concentration of
ions in the SBF has been investigated with AAS spectroscopy.
Release of Ag ions from all the samples to the solution as a
function of time and square root of time is given in Fig. 6(a) and
(b) respectively. Release of Ag ions has been checked up to 120 h.
As shown in the Fig. 6(a), release of Ag ions from the samples is
related to the content of the silver. It has been observed that when
the content of Ag increases, release concentration also increases.
Values of R2
calculated for controlled release of Ag for all the
samples are shown in Fig. 6(b). All the values are higher than 0.95
which indicate that Ag releasing data is perfectly fitted as a
function of square root of time. Therefore, it can be inferred that
extraction of Ag ions from the samples follow a diffusion con-
trolled mechanism. Controlled release of silver can lead to long
lasting antimicrobial properties and also, protect the surrounding
tissues from the toxicity of silver.
3.3. Drug release studies
In-vitro drug release of prepared samples has been studied by
using gentamycin as an antibiotic. Details of experiment procedure
are reported by authors elsewhere [29]. Briefly 1 g of the sample
has been added into 50 mL of gentamycin solution. Mixture has
been stirred for 24 h. followed by incubation of 30 h. at 37 °C.
Release of drug in the SBF solution has been investigated with the
help of UV-Visible spectroscopy at 243 nm.
Fig. 7(a) represent the concentration of drug released in the
SBF. It can be observed that all the prepared samples have shown
similar drug release properties. Approximately 80 to 90% drug
release has been observed within 30 h. Almost 70% drug has been
released within the first 6 h. and in next 14 h. drug released has
been observed to increase up to 15%. Further, a small increase (up
to 5%) has been observed. Different rates of drug release can be
explained on the basis of encapsulation of drug molecules in si-
lanol groups of nanostructures by interaction between functional
group of drug molecule and present silanol group [49] and also in
the pores of sample. Fast release rate can be related to the release
of drug molecule from the pores of the sample. On the other hand,
slow release rate is speculated to be due to the high zeta potential
property of nano structure, which holds the drug molecule tightly.
Change in the pore size and surface area of sample during drug
release have been investigated with the help of BET study (Fig. 7
(b)). Table 1 provides the quantitative change in the pore volume
and surface area of sample. Pore size and surface area decrease
due to encapsulation of drug into the pores by interaction between
functional group of drug molecules and silanol group. Drug en-
capsulated samples have been immersed in SBF for 30 h. Release of
drug molecules from the pores of the sample is identified by in-
crease in the surface area and pore size.
3.4. Antimicrobial studies
Tendency to kill the microorganisms of samples has been stu-
died with different gram negative and gram positive micro-
organisms. The reference strains included Gram positive bacteria
Staphylococcus aureus (MTCC740), Gram negative bacteria, Kleb-
siella pneumoniae (MTCC109), Shigella flexneri (MTCC1457), Sal-
monella typhimurium (MTCC 1251) and Escherichia coli (MTCC-
119). These were obtained from Microbial Type Culture Collection
(MTCC), Institute of Microbial Technology (IMTECH), Chandigarh,
India. Clinical isolate Methicillin Resistant Staphylococcus aureus
(MRSA) has been obtained from Post Graduate Institute of Medical
Education and Research, (PGIMER), Chandigarh, India. The bac-
terial cultures were maintained on nutrient agar slants. A loopful
of isolated bacterial colonies were inoculated into 5 mL of nutrient
broth and incubated at 37 °C for 4 h. The turbidity of actively
growing bacterial suspension was adjusted to match the turbidity
standard of 0.5 Mc Farland units prepared by mixing 0.5 mL of
Table 2
Observed FTIR peaks as a function of wavenumber.
Wavenumber
(cmÀ1
)
Structural units References
Near 460 Si-O-Si bending [38]
522 Si-O bending [43]
528 Si-O bending [43]
559 Vibrations of −PO4
3 bond [42]
601 Vibrations of −PO4
3 bond [42]
Near 801 Repolymerization of Si-O-Si [38]
Near 878 Vibrations of − −SiO and SiO4
4
7
4 and acidic
phosphate group
[38]
1044–1080 vibrations of Antisymmetrical Si-O-Si bond
and ionic character of −PO4
3 bond
[38,44,45]
∼1385 Vibrations of −CO3
2 group [46]
1420–1530 Vibrations of C-O bond [42]
∼1640 O-H bending vibrations [46]
Table 3(d)
XRD hkl data
(Combeite,
78-1650).
2θ° hkl
26.9 211
29.6 300
33.6 024
34.3 220
42.2 042

1.75% (w/v) barium chloride dihydrate (BaCl2 Á 2H2O) to 99.5 mL of
0.18 M (v/v) sulphuric acid with constant stirring. The bacterial
suspension prepared has been used for testing their sensitivity to
all the compounds (sample dissolved in dimethyl sulfoxide i. e.
DMSO). All the compounds containing silver have been found to be
active against almost all the microorganisms tested. S. aureus and
K. pneumoniae are found to be the most sensitive organism and the
large and more obvious inhibition zones are found to be in the
range of 21 mm to 10 mm as shown in Fig. 8(a). Gram negative
bacteria acquire resistance more readily due to their outer mem-
brane which contains narrow pore in channels which retard the
entry into the cell, of even small hydrophilic compounds, a lipo-
polysaccharide moiety which slows down the trans-membrane
diffusion of lipophilic antibiotics and they often possess a multi-
drug efﬂux pump which eliminates many antibiotics from the cells
causing several diseases. All the Ag and Mg containing samples
have shown good antimicrobial potential against K. pneumoniae, S.
ﬂexneri and S. typhimurium with zone of inhibition ranging from
20 to 21 mm, 10–13 mm and 10–15 mm respectively. The im-
portance of the reported study became paramount when resistant
strains like MRSA were also found sensitive to the prepared sam-
ples. MRSA is a type of staphylococcus bacteria. It is not found in
the natural environment. This lives on nose and skin of humans.
MRSA can cause life threatening infection and is encountered
specially in hospitals, prisons and nursing homes. Due to antibiotic
resistant form of Staphylococcus aureus, it is a challenging bacter-
ium in clinical medicine. Zone of inhibition ranging from 12 to
15 mm has been obtained with MRSA on the prepared samples
(Fig. 8(b)).
MIC values have been obtained by agar dilution method. A
Fig. 5. (a) Weight loss trend, (b) pH graph, (c) Concentration of different ions in citric buffer, (d) Concentration of different ions in SBF (e) XRD spectra of samples treated
with citric buffer shows the formation of Â Calcium Silicate and ▲ Silver. Error bars for 5 (a)–(d) indicate the standard deviation observed for three measurements.

stock solution of all the compounds 2.5% (25 mg/mL) concentra-
tion were prepared and incorporated into Muller Hinton agar
medium. The ﬁnal concentrations of the compounds in the med-
ium containing plates have the range of 0.05–5 mg/mL. These
plates were then inoculated with 10 mL of the activated bacterial
strains by streaking with a sterile tooth pick. The plates were in-
cubated at 37 °C for 24 h. The lowest concentration of the sample
causing complete inhibition of the bacterial growth was taken as
MIC. The results were compared with that of control in which the
sample was replaced with DMSO. MIC values against S. aureus and
K. pneumoniae has been checked and found 0.75 mg/mL for silver
containing samples. Results have shown that our samples can be
potent antimicrobial agents which can be further exploited for
various pharmaceutical processes like wound healing and dental
clinical applications.
3.5. Cytotoxicity and cell culture study
In vitro cellular toxicity of butanolic extract and the puriﬁed
compounds has been determined by MTT (3-[4,5-dimethylthiazol-
2-yl]-2, 5-diphenyl tetrazolium bromide) assay. 10 mL sheep blood
has been taken into injection syringe containing 3 mL Alsever's
solution (anticoagulant) and transferred to sterile centrifuge tubes.
The blood has been centrifuged at 1600 rpm at room temperature
for 20 min to separate the plasma from the cells. The supernatant
has been discarded and centrifuged again adding 6 mL phosphate
buffer saline (PBS). The blood cells have been washed thrice with
PBS by centrifugation and the pellets have 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 a
light microscope so as to obtain cells equivalent to 1 Â 105
cells.
The following formula has been used to determine the required
number of cells:
=
× × ( )
−Number of cells mL Number of cells counted in 25
squares Dilution factor 10 4
1
4
The cell suspension thus prepared is dispensed into Elisa plates
(100 mL /well) and incubated at 37 °C for overnight. The super-
natant has been removed carefully and 200 mL of the compound is
added and incubated further for 24 h. Supernatant has been re-
moved again and 20 mL MTT solutions (5 mg/mL) has been added
to each well and incubated further for 3 h. at 37 °C on orbital
shaker at 60 rpm. After incubation, the supernatant has been re-
moved without disturbing the cells and 50 mL DMSO has been
added to each well to dissolve the formazan crystals. The absor-
bance has been measured at 590 nm using an automated micro
Fig. 6. Change of concentration of silver ion released in SBF (a) with time and (b) with square root of time. Error bar in (a) indicates the standard deviation observed for three
measurements.
Fig. 7. (a) Drug release study in SBF and (b) Quantity absorbed as function of relative pressure during BET analysis. Error bar in (a) indicates the standard deviation observed
for three measurements.

plate reader (Biorad 680-XR, Japan). The wells with untreated cells
served as control.
Behavior of the samples on human osteosarcoma cell line (MG
63) has also been examined. The MG 63 human osteosarcoma cell
line has been procured from the National Center for Cell Science,
Pune, India. The culture has been maintained in DMEM (Dulbec-
co's Modified Eagle's Medium) supplemented with 10% heat in-
activated FBS (fetal bovine serum), glutamine (20 mM), strepto-
mycin (100 mg mLÀ1
) and gentamycin (100 mg mLÀ1
) at 37 °C and
humid environment with 5% CO2. The MTT (3-[4, 5-di-
methylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) dye has
been used to check the cell integrity. The MG-63 cells
(2 Â 104
cell mLÀ1
) have been incubated with different samples in
24 well tissue culture plates for 96 h. Tissue culture grade cover-
slip (Himedia) has been used as a positive control for comparison.
Each sample has been tested in triplicate. Afterwards, the cells
have been incubated with 500 mL MTT dye after 92 h. incubation
(0.5 mg mLÀ1
) in a non-serum medium for 4 h. resulting in a blue
coloured formazan crystal formation. These formazan crystals
have been solubilized with 500 mL of DMSO and read at 570 nm
using Labsystem Multiskan EX ELISA reader.
Fig. 9 represents cell viability for the purpose of cytotoxicity
study. It has been observed that all the samples are non-toxic in
nature with more than 80% cell viability. In-vitro cell proliferation
has also been checked with MG-63 osteoblast cell lines. FESEM
micrograph of MG-63 cell growth on the surface of the sample has
been provided in Fig. 10(a). All the samples have shown more than
80% cell proliferation (Fig.10(b)), whereas the sample BMA-0 has
shown 12% more growth of MG-63 cell lines as compared to
control. It has been observed that the samples containing Mg and
Ag reported low cell proliferation as compared to Mg and Ag free
samples. It may be due to toxic nature of Ag which slows down the
proliferation rate of MG-63 cell lines. All the prepared samples
have provided a friendly environment for the growth of MG-63
cell lines.
Many authors have studied the structural and bioactive prop-
erties of Mg [18,29,30,41,50] or Ag [9,22,27] individually within
the bioactive systems. As per literature [8,34,51,52], formation of
the nanostructure enhance the bioactivity of the samples. Prabhu
et. al.[53] have reported the bioactivity of Mg containing nano
bioactive glass system prepared by quick alkali- mediated sol gel
method after 21 days of in vitro study. They also reported non-
reactivity of samples against microorganisms S. aureus and E. coli.
Their samples have not shown any zone of inhibition against both
microorganisms. Goh et al. [9] also have prepared silver containing
nano bioactive glass particles by same procedure. They reported
the bioactivity of the samples after 3 days of in vitro study. They
also reported the activity of the samples against one micro-
organism (E. coli) only. Jha and Singh [54] have observed that the
replacement of CaO by MgO retard bioactive properties of the
55SiO2-10K2O-(35Àx) CaO-xMgO (x¼5, 15, 25 and 35) glass sys-
tem. This result is consistent with the speculation of Pedone,
Malavasi and Menziani [55]. Similarly, authors have also observed
slower bioactivity with the addition of Mg. Bioceramics can be
successful implant materials if they have higher values for cell
viability. In the synthesized samples, it has been found that the
addition of magnesium oxide from 2 to 10 mol% has improved the
cell viability from 86% to 97%. Therefore, it can be concluded that
although Mg has retarded the bioactivity of the synthesized bio-
ceramics but it has significantly enhanced the cell viability of the
samples. Moreover, Mg can be used to control the degradation rate
[15–17], maintenance of polymerized silica network and improve
the osteoporosis and metabolic syndrome. Therefore, Mg can play
a vital role in the composition of the successful implant bioceramic
materials and hence, the study of the effect of the inclusion of
magnesium on the properties of bioceramics can be useful in-
vestigation. Due to these reasons, authors have reported the in-
fluence of both Mg and Ag together on the structural, biological,
drug delivery and degradation properties of bioceramic samples.
Prepared samples have the ability to overcome the risk of lack of
Fig. 8. (a) Bar graphs of different microbial activity with samples and (b) Representative figure for microbial activity. Error bar in (a) indicates the standard deviation
observed for three measurements.
Fig. 9. Cell viability of sample during cytotoxicity test. Error bar indicates the
standard deviation observed for three measurements.

tissue integration and infection which are two frequent problems
associated with implant materials in orthopaedic and dental areas.
The particular aim of this work is to study the controlled release of
silver which leads to excellent antibacterial activities against six
different gram þve and gram –ve microorganisms including
highly resistive strain like MRSA. Our samples have shown bioac-
tivity after one day of in vitro study which is much faster as
compared to the report by Prabhu et al. [53] and Goh et al. [9].
Authors speculate that the appropriate ion release speed of dif-
ferent elements (Ca, Na, P, Si, Mg and Ag) has contributed to the
enhanced bioactivity. This feature can accelerate the filling of bone
defects by improving the time for repair of damaged cortical bone.
Therefore, our new prepared bioactive ceramic compositions may
have significant role in the area of bioactive implant materials,
especially, in terms of quicker growth of HAp layer and better
antimicrobial properties. Results indicate that Mg and Ag con-
taining bioceramics have the potential for use in clinical
applications.
4. Conclusions
All samples have been found to be crystalline in nature with
combeite as a major phase in Mg and Ag free samples. Growth of
hydroxyl apatite layer on all samples has occurred within 1 day in
SBF demonstrating the excellent bioactive behavior of the syn-
thesized compositions. Degradation and ion release properties of
these samples have been found to be composition dependent.
Moreover, samples have been estimated to be good host materials
for antibiotic drugs due to specific values for pore size and surface
area. The microbial assay revealed that Ag enhances the anti-
bacterial properties against different microorganisms. This could
validate their ability to treat bone infection. Controlled release of
Ag is very important for friendly environment of cell growth
without abrupt change in pH values. In Mg and Ag containing
samples, BMA-5 may be considered as the best sample due to good
degradation rate, highly porous nature, more than 90% cell viabi-
lity and excellent efficiency to kill the microorganisms. Our pre-
pared compositions have the potential to repair the damaged bone
within short interval of time and also, to provide the protection
against the infection from different microorganisms.
Acknowledgments
The authors Kulwinder Kaur and Vikas Anand are grateful to
the financial assistance provided by the DST, New Delhi (India)
through INSPIRE program SRF[IF-120620] and UGC, New Delhi
(India) through SRF[F. 17–74/2008(SA-I)] respectively.
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