Journal of Materials Science:
Materials in Medicine
Official Journal of the European Society
J Mater Sci: Mater Med (2015) 26:1-12
Investigating the influence of Na+ and Sr2+
on the structure and solubility of SiO2–
TiO2–CaO–Na2O/SrO bioactive glass
Y. Li, L. M. Placek, A. Coughlan,
F. R. Laffir, D. Pradhan, N. P. Mellott &
A. W. Wren
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BIOMATERIALS SYNTHESIS AND CHARACTERIZATION
Investigating the inﬂuence of Na+
on the structure
and solubility of SiO2–TiO2–CaO–Na2O/SrO bioactive glass
Y. Li • L. M. Placek • A. Coughlan •
F. R. Lafﬁr • D. Pradhan • N. P. Mellott •
A. W. Wren
Received: 3 July 2014 / Accepted: 1 November 2014
Ó Springer Science+Business Media New York 2015
Abstract This study was conducted to determine the
inﬂuence that network modiﬁers, sodium (Na?
) and stron-
), have on the solubility of a SiO2–TiO2–CaO–
Na2O/SrO bioactive glass. Glass characterization deter-
mined each composition had a similar structure, i.e. bridging
to non-bridging oxygen ratio determined by X-ray photo-
electron spectroscopy. Magic angle spinning nuclear mag-
netic resonance (MAS-NMR) conﬁrmed structural
similarities as each glass presented spectral shifts between
-84 and -85 ppm. Differential thermal analysis and hard-
ness testing revealed higher glass transition temperatures (Tg
591–760 °C) and hardness values (2.4–6.1 GPa) for the
containing glasses. Additionally the Sr2?
containing glasses displayed much lower ion release rates
than the Na?
(*1,200 mg/L) containing glass analogues.
With the reduction in ion release there was an associated
reduction in solution pH. Cytotoxicity and cell adhesion
studies were conducted using MC3T3 Osteoblasts. Each
glass did not signiﬁcantly reduce cell numbers and osteo-
blasts were found to adhere to each glass surface.
Bioactive glasses have generated considerable interest in the
recent past as a medical material. Since the inception of
in the late 1960s by Prof Larry Hench, numerous
glass compositions have been investigated for their thera-
peutic potential . The original composition (45S5 Bio-
), is composed of 45 % SiO2–24.5 % Na2O–24.5 %
CaO–6 % P2O5 and it was determined that when implanted as
cast glass blocks in a rat femoral implant model, the glass
blocks bonded to the surrounding bone [2–4]. Since this
discovery, many formulations of bioactive glass have been
investigated from a structural aspect to determine their solu-
bility, degradability and subsequent therapeutic effect in vivo
. Many commercial materials have resulted from this class
of materials including bulk implants to replace bones or teeth,
coatings to anchor orthopedic or dental devices, or in the form
of powders as bone grafts to ﬁll defects in bone [3, 5]. Glass
compositions such as BioglassÒ
have the highest rates of
bioactivity and lead to rapid regeneration of trabecular bone
with a composition, architecture and quality that matches the
host tissue. The regeneration of bone is due to a combination
of processes; termed osteostimulation and osteoconduction
. In particular, these reactions involve dissolution of criti-
cal concentrations of soluble Si4?
ions that gives
rise to both intracellular and extracellular responses at the
interface of the glass with its physiological environment .
These responses result in the rapid formation of osteoid
bridges between particles followed by mineralization to
produce mature bone structures [3, 4].
The dissolution and subsequent ion release from these
materials is known to be the predominant characteristic that
initiates the mineralization process as network modiﬁers
) from the glass react with H?
(H3O) ions from the
solution leads to hydrolysis of the silica groups with the
Y. Li Á L. M. Placek Á D. Pradhan Á N. P. Mellott Á
A. W. Wren (&)
Inamori School of Engineering, Alfred University, Alfred,
NY 14802, USA
School of Materials Engineering, Purdue University,
West Lafayette, IN, USA
F. R. Lafﬁr
Materials and Surface Science Institute, University of Limerick,
J Mater Sci: Mater Med (2015) 26:85
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creation of Silanol (Si–OH) [1, 4, 7]. Condensation of an
amorphous Si-rich layer (depleted in Ca2?
ceeds on the glass surface followed by migration of Ca2?
ions from the glass through the Si-rich layer leading to
the formation of an amorphous CaP (ACP) surface layer.
Over time the ACP surface layer incorporates ions such as
from the surrounding environment which
crystallizes to hydroxyapatite [4, 8]. However, a complication
that can contribute to local toxicity in vivo is due to the sol-
ubility of these ions and the degradation rate of the glass. By
increasing the concentration of ions such as Na?
the glass, local environmental changes can occur, in particular
the pH. The biological effects of these changes are difﬁcult to
predict and their biological role, toxicity, and removal has not
been clearly determined [4, 6].
It is understood that the introduction of network modiﬁers
) within the glass can lead to the disruption or
the breaking of Si–O–Si bonds within the SiOx tetrahedrons,
leading to the development of non-bridging oxygen species
). It is understood that the dissolution and deg-
radation of bioactive glasses are directly related to the con-
centration of NBOs within the glass structure, and this is in
turn related to the concentration of alkali and alkali earth
cations [6, 9]. While studies have been conducted to investi-
gate the precise role that network formers contribute to a glass
structure [10, 11], this study aims to determine the effect that a
) and a divalent (Sr2?
) cation can have on the
structure of a bioactive glass, and the subsequent solubility.
was selected as the monovalent cation as its role in bio-
active glasses has been well described [4, 7], and Sr2?
known to have positive therapeutic effects in vivo, where it has
been cited as increasing the proliferation of osteoblasts while
reducing osteoclastic activity . This coupling activity has
resulted in the development of an anti-osteoporotic drug,
strontium ranelate that is used to increase bone mineral density
in patients with metabolic bone diseases such as osteoporosis
[12, 13]. It is known that both of these cations (Na?
predominantly as a network modiﬁer within a glass structure
[3, 14], and this study aims to use complementary character-
ization techniques such as high resolution XPS, magic-angle
spinning nuclear magnetic resonance (MAS-NMR) and
Raman spectroscopy to investigate any structural differences
within the glass as a result of Na?
subsequent effect on bioactivity, speciﬁcally cell viability and
cell adhesion will be investigated using MC3T3 Osteoblasts.
2 Materials and methods
2.1 Glass synthesis
Three glass compositions (Ly-N, Ly-C, Ly-S) were formu-
lated for this study with the principal aim being to
investigate structural and solubility changes within a bio-
active glass as a function of Sodium (Na?
, Ly-N) and
, Ly-S) incorporation. A control glass (Ly-
C) was also formulated which contained equal quantities of
. Glasses were prepared by weighing out
appropriate amounts of analytical grade reagents and ball
milling (1 h). Different glass samples were produced for
testing throughout this study and are explained as follows.
2.1.1 Glass powder production
The powdered mixes were oven dried (100 °C, 1 h) and
ﬁred (1,500 °C, 1 h) in a platinum crucible and shock
quenched in water. The resulting frits were dried, ground
and sieved to retrieve glass powders with a particle size of
45 lm (XRD, DTA, Raman, MAS-NMR, pH, ICP).
2.1.2 Glass rod production
The powdered mixes were oven dried (100 °C, 1 h) and ﬁred
(1,500 °C, 1 h) in platinum crucibles. Glass castings were
produced by pouring the glass melts into graphite molds
which were preheated to Tg. The graphite molds were left for
3 h and furnace cooled in order to anneal the glass. The
resulting glass casts were then cut with a diamond blade on an
Isomet 5000 Linear Precision Saw (1,500 rpm, 0.4 mm/min)
and were shaped into rods of 15 9 3 mm using a Phoenix
4000 grinding machine with 60 lm silicon carbide grinding
paper, Buehler, IL, USA (High resolution XPS).
2.1.3 Glass plate production
The powdered mixes were oven dried (100 °C, 1 h) and
ﬁred (1,500 °C, 1 h) in platinum crucibles. Glass plates
measuring[18 mm in diameter were produced by pouring
molten glass on a graphite plate that was pre-heated to the
samples Tg. The glass plates were then annealed for 3 h
and furnace cooled (XRF, Hardness).
2.1.4 Glass button production
The powdered mixes were oven dried (100 °C, 1 h) and
ﬁred (1,500 °C, 1 h) in platinum crucibles. Glass buttons
were produced by drilling holes (8 mm) in a ﬂat graphite
plate measuring 4 mm in thickness. This mold was placed
on another ﬂat graphite plate and heated to the individual
samples Tg. Molten glass was poured into each button mold
and pressed to form an approximately uniform 8 9 4 mm
button. Each button was annealed for 3 h and furnace
cooled, and then ground and polished using 60 lm silicon
carbide grinding paper (Buehler, IL, USA). Final glass
buttons measuring 8 9 2 mm were polished further to a
ﬁne surface and ultrasonically cleaned and autoclaved. 6
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buttons were produced per glass composition for cell cul-
ture testing (Cytotoxicity, Cell Adhesion).
2.2 Glass characterization
2.2.1 X-ray ﬂuorescence (XRF)
X-ray ﬂuorescence was undertaken using the S4 Pioneer
(Bruker AXS Inc, MA, USA) to calculate the chemical
composition of each glass. Glass plates ([18 mm in
diameter) were placed in a holder with an 18 mm mask
(thus revealing 18 mm diameter of the glass for testing)
and underwent testing using the MultiVac 18 program. The
results were quantiﬁed using the Spectra Plus Launcher
(Bruker) and normalized to 100.
2.2.2 Network connectivity (NC)
The network connectivity (NC) of the glasses was calcu-
lated with Eq. 1 using the molar compositions of the glass.
Network connectivity calculations were performed
assuming that Ti performs as a network former and also as
a network modiﬁer as Ti is a known network intermediate.
No:BOs À No:NBOs
Total No: Bridging Species
where: NC = network connectivity, BO = bridging oxy-
gens, NBO = non-bridging oxygens.
2.2.3 X-ray diffraction (XRD)
Diffraction patterns were collected using a Siemens D5000
X-ray diffraction unit (Bruker AXS Inc., WI, USA). Glass
powder samples were packed into standard stainless steel
sample holders. A generator voltage of 40 kV and a tube
current of 30 mAwas employed. Diffractograms were
collected in the range 10° 2h 70°, at a scan step size
0.02° and a step time of 10 s.
2.2.4 Differential thermal analysis (DTA)
A combined differential thermal analyzer-thermal gravimet-
ric analyzer (DTA-TGA) (Stanton Redcroft STA 1640,
Rheometric Scientiﬁc, Epsom, UK) was used to measure the
glass transition temperature (Tg) for all glasses. A heating rate
of 10 °C/min was employed using a nitrogen atmosphere with
an alumina crucible where a matched alumina crucible was
used as a reference. Sample measurements were carried out
every 6 s between 30 and 1,300 °C.
2.2.5 X-ray photoelectron spectroscopy (XPS)
High resolution XPS was performed in a Kratos AXIS 165
spectrometer (Kratos Analytical, Manchester, UK) using
monochromatic Al Ka radiation (ht = 1,486.6 eV). Glass
rods with dimensions of 15 9 3 9 3 mm were produced
from the melt and fractured under vacuum (*2 9 10-8
torr) to create pristine surfaces with minimum contamina-
tion. Surface charging was minimized by ﬂooding the
surface with low energy electrons. The C 1 s peak of
adventitious carbon at 284.8 eV was used as a charge
reference to calibrate the binding energies. High resolution
spectra were taken at pass energy of 20 eV, with step size
of 0.05 eV and 100 ms dwell time. For peak ﬁtting, a
mixed Gaussian-Lorenzian function with a Shirely type
background subtraction was used.
2.2.6 Raman spectroscopy
Raman analysis was performed on a Witec Confocal
Raman Microscope CRM200 equipped with Si detectors,
green laser with an excitation wavelength of 532 nm and
power of 70 mW, and a dispersion grating selected of
600 L/mm. The instrument was calibrated using standard
silicon, including a test run on a focus spectrum. This was
performed to optimize the intensity of the beam. The
characteristic Si line at 520 cm-1
was maximized through
optimization of SMA connector.
2.2.7 Magic angle spinning-nuclear magnetic resonance
SiMAS NMR spectra were recorded using a 14 T (tesla)
Bruker Advance III wide-bore FT-NMR spectrometer
(Billerica, MA, USA), equipped with a double broadband
tunable triple resonance HXY CP-MAS probe. The glass
samples were placed in a zirconia sample rotor with a
diameter of 4 mm. The sample spinning speed at the magic
angle to the external magnetic ﬁeld was 10 kHz. 29
NMR spectra were acquired at 300 K with the transmitter
set to *119.26 MHz (-100 ppm) with a 3.0 us pulse
length (pulse angle, p/2), 120-s recycle delays, where the
signals from 640 scans were accumulated for Ly-S, Ly-C,
and Ly-N, respectively. 29
Si NMR chemical shifts are
reported in ppm, with TMSP (trimethylsilylpropionate) as
the external reference (0 ppm). Data were processed using
a 25 Hz Gaussian apodization function followed by base-
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2.3 Investigating glass solubility
2.3.1 Particle size analysis (PSA)
Multisizer 4 Particle Size Analyzer (Beckman- Coulter,
Fullerton, CA, USA). The glass powder samples were eval-
uated in the range of 0.4–100.0 lm and the run length took
60 s. The ﬂuid used was water and was used at a temperature
range between 10 and 37 °C. The relevant volume statistics
were calculated on each glass (where n = 3/sample).
2.3.2 Advanced surface area and porosity (ASAP)
In order to determine the surface area of each glass
Advanced Surface Area and Porosimetry, Micromeritics
ASAP 2020 (Micrometrics Instrument Corporation, Nor-
cross, USA) was employed. Approximately 60 mg of each
glass sample (Ly-N, Ly-C and Ly-S) was analyzed and the
speciﬁc surface area was calculated using the Brunauer-
Emmett-Teller (BET) method, (where n = 3/sample).
2.3.3 Ion release proﬁles (ICP)
Glass powders (Ly-N, Ly-C and Ly-S, where n = 3/compo-
sition) were incubated in 10 mL of sterile de-ionized water
with surface areas of 1 m2
for 1, 7, 14 and 21 days. The
sterile DI water was exchanged after each time period to
determine if depletion in ion release occurs as the glasses
incubation time increases. Sample tubes were centrifuged
(3,000 rpm, 5 min) prior to removing the ﬂuids, and dried for
12 h in an incubator at 37 °C. Then 10 mL sterile DI water
was added to the glass samples and stored on a rotary mixer
until the next time period where the process was repeated. All
ﬂuids extracted (at 1, 7, 14, 21 days, n = 3) were stored in a
fridge until testing and were used for ion release and pH
testing at each time period. Concentrations of Sodium (Na?
), Titanium (Ti4?
), Calcium (Ca2?
) were determined using Inductively Cou-
pled Plasma-Atomic Emission Spectroscopy (ICP-AES) on
a Perkin-Elmer Optima 5300UV (Perkin Elmer, MA, USA).
ICP-AES calibration standards for Ca, Si, Ti and Na/Sr ions
were prepared from a stock solution on a gravimetric basis.
Three target calibration standards were prepared for each ion
and de-ionized water was used as a control.
2.3.4 pH analysis
Changes in pH of the ICP solutions were monitored using a
Corning 430 pH meter after 1, 7, 14 and 21 days incuba-
tion. Prior to testing, the pH meter was calibrated using pH
buffer solution 4.00 ± 0.02 (Fisher Scientiﬁc, Pittsburgh,
PA). Measurements were recorded in triplicate and De-
ionized water (pH 7.0) was used as a control and was
measured at each time period.
2.4 Hardness testing
Hardness testing was completed on glass plates mounted in
epoxy resin. A total of 10 measurements were taken on
each glass plate and 3 regions on the each glass plate were
analyzed (total n = 30/sample). A Shimadzu HMV-2000
Hardness testing machine was used with a 500 g load cell
with 15 s intervals.
2.5 Cell culture analysis
2.5.1 Cytotoxicity analysis
MC-3T3-E1 Osteoblasts (ATCC CRL-2593) were used for
this study and were maintained on a regular feeding regime
in a cell culture incubator at 37 °C/5 % CO2/95 % air
atmosphere. Cells were seeded into 24 well plates at a
density of 20,000 cells per well and incubated for 24 h
prior to testing. The culture media used was Minimum
Essential Medium Alpha Media supplement with 10 %
fetal bovine serum and 1 % (2 mM) L-glutamine (Camb-
rex, MD, USA). Cell culture analysis was conducted using
glass buttons as prepared in Sect. 2.1.4. Glass buttons were
incubated in 24 well plates for 24 and 48 h in Minimum
Essential Medium Alpha Media (n = 3/sample/time per-
iod). For cell viability testing, 100 lL of liquid extract was
removed (n = 3 per sample well) and these liquid extracts
were used for cytotoxicity testing using the Methyl Tetra-
zolium (MTT) assay. Extracts (100 lL) of sample (Ly-N,
Ly-C and Ly-S at 24 h and 48 h) were added into wells
containing MC-3T3-E1 Osteoblasts in culture medium
(1 mL) and the 24 well test plates were then incubated
for 24 h at 37 °C/5 % CO2. The MTT was added in an
amount equal to 10 % of the culture medium volume/well.
The cultures were then re-incubated for a further 2 h
(37 °C/5 % CO2) after which, the cultures were removed
from the incubator and the resultant formazan crystals were
dissolved by adding an amount of MTT Solubilization
Solution (10 % Triton x-100 in Acidic Isopropanol (0.1 n
HCI)) equal to the original culture medium volume. Once the
crystals were fully dissolved, the absorbance was measured
at a wavelength of 570 nm. Control media and healthy
growing cell population (n = 3) were used as a reference.
2.5.2 Osteoblast adhesion procedure
The MC3T3-E1 osteoblast cells were cultured as explained
in Sect. 2.5.1. After 48 h incubation, media was removed
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and 5 mL trypsin was added to the culture ﬂask. The cells
were left to detach for 20 min, after this time, trypsin was
removed (centrifuge, 1,500 rpm, 5 min) and cells were re-
suspended in culture media. The trypsin was removed and
10 mL media was added. The number of cells was calcu-
lated to 20,000 cells per/ml media. The glass buttons were
placed in each well where 1 mL cell/media solution was
seeded onto the surface of the glass buttons and incubated
for 24 h (n = 3 per composition) and 48 h (n = 3 per
composition). Glass buttons were extracted after 24 and
48 h and were ﬁxed with 4 % (w/v) paraformaldehyde in
1* PBS buffer for 30 min, and then post-ﬁxed with 1 %
osmium tetroxide in distilled water for 1 h. Samples were
dehydrated with a series of graded ethanol washes (50/60/
70/80/90/100 % DI water). Samples were immersed in
hexamethyldislizane for 5 min and then transferred to a
desiccator for 30 min. The glass plates were then coated in
gold and sample imaging was carried out using an FEI Co.
Quanta 200F Environmental Scanning Electron Micro-
scope equipped with an EDAX Genesis Energy-Dispersive
2.6 Statistical analysis
One-way analysis of variance (ANOVA) was employed to
compare the difference in hardness as a function of com-
position. Additionally, differences in cell viability was
evaluated based on sample composition compared to the
healthy growing cell population at both 24 and 48 h.
Comparison of relevant means was performed using the
post hoc Bonferroni test. Differences between groups was
deemed signiﬁcant when P B 0.05.
3.1 Glass characterization
A bioactive glass series was produced to investigate the
effect that Sodium (Na?
) and Strontium (Sr2?
) have on the
glass structure, solubility and subsequent bioactivity.
containing glass (Ly-N), an intermediate glass
containing both Na?
(Ly-C), and a Sr2?
taining glass (Ly-S) were synthesized for this study. Initial
characterization techniques included X-ray diffraction
(XRD) to conﬁrm the amorphous nature of each of the
starting glasses (Fig. 1a) while differential thermal analysis
(DTA) was used to identify the thermal characteristics of
Ly-N, Ly-C and Ly-S. Figure 1b shows the DTA proﬁle for
Ly-N, Ly-C and Ly-S. Regarding Ly-N, the glass transition
temperature (Tg) was found to be 591 °C while a small
endotherm was present at approximately 700 °C with the
predominant crystallization peak (Tc1) being at 777 °C. For
Ly-C the Tg was 650 °C while Tc1was present at 778 °C.
For Ly-S the Tg was considerably higher than both Ly-
N and Ly-C at 760 °C while Tc1 was evident at 871 °C. The
network connectivity (NC) of each glass was calculated
using the original batch calculations and the composition
determined by X-ray ﬂuorescence (XRF, Fig. 2a). XRF
data (Table 1) determined that the original batch compo-
sitions are comparable to the XRF determined composi-
tions. NC calculations were conducted as they are a
theoretical method of determining the connectivity of the
Si–O–Si bonds within a glass and were performed assum-
ing that Titanium (Ti4?
) acts as both a network former and
also as a network modiﬁer. Assuming Ti4?
acts as a net-
work modiﬁer, the theoretical calculation predicts a NC of
2.36 for each glass, while XRF data predicts a NC of 2.26
for Ly-N and Ly-C and a NC of 2.42 for Ly-S. This dif-
ference is due to the slightly higher Si4?
determined by XRF. Assuming Ti4?
acts as a network
former the NC is calculated to be 2.67, while XRF data
predicts a NC of 2.58 for Ly-N and Ly-C, and 2.72 for Ly-
S. Hardness testing is presented in Fig. 2b for each glass
which shows the Sr2?
containing glasses to have signiﬁ-
cantly higher hardness values (Ly-C at 6.01GPa, Ly-S at 5.5
GPa) than the Na?
glass (Ly-N at 2.2 GPa).
Characterization techniques for analyzing glass struc-
ture, such as X-ray photoelectron spectroscopy (XPS),
Raman spectroscopy and magic angle spinning nuclear
magnetic resonance (MAS-NMR) were employed to
determine if any signiﬁcant differences in glass structure
were evident as a result of Na?
resolution X-ray photoelectron spectroscopy (XPS) was
conducted on each glass, where the O1s signal is presented
in Fig. 3. Figure 3a shows the high resolution O 1s of Ly-
N where the spectra was resolved to reveal two peaks at
binding energies (B.E.) of 529.7 and 531.3 eV which are
representative of the non-bridging oxygen (NBO) and
bridging oxygen (BO) concentration respectively. Ly-
C presented peaks that were slightly shifted to lower
binding energies of 529.9 eV (NBO) and 531.6 eV (BO)
while Ly-S experienced a similar shift to 530.1 eV (NBO)
and 531.8 eV (BO). Irrespective of composition, the ratio
of BO/NBO was consistent as 45:55 suggesting that both
assume a similar role (network modiﬁer) in
the glass series. High resolution XPS was also conducted
on each element and the results are presented in Table 2.
Regarding Si 2p there was a slight shift in B.E. from
101.5 eV (Ly-N) to 101.8 eV (Ly-C) and 102.1 eV (Ly-S).
High resolution scans of Ca 2p and Ti 2p experienced
similar shifts in B.E. from a lower B.E. in Ly-N to a higher
B.E. evident in Ly-S. With respect to the Na?
glasses the Na 1s peak shifted from 1,070.6 eV (Ly-N) to
1,071.2 eV (Ly-C). The Sr2?
containing glasses also
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experienced a slight shift from 133.2 eV (Ly-C) to
133.5 eV (Ly-S).
Raman spectroscopy was conducted on each of the
glasses and the resulting spectra are presented in Fig. 4. It
is evident from Fig. 4 that the spectra presented similar
characteristics for each glass, particularly at lower wave-
numbers. Each glass, Ly-N, Ly-C and Ly-S present a similar
band at 344 cm-1
and also at approximately 605 cm-1
slight shift in wavenumbers was observed within the region
of 800–1,000 cm-1
. Ly-N (Fig. 4a) presented a peak at
within a relatively narrow spectral region
between 900 and 1,000 cm-1
when compared to Ly-C and
Ly-S. Ly-C (Fig. 4b) presented a broad absorption band at
which shifted to lower wavenumbers, 852 cm-1
for Ly-S (Fig. 4c) with further broadening of the spectral
envelope ranging from 900 to 1,000 cm-1
. An additional
peak was observed for each of the glasses which ranged
between 1,052 and 1,060 cm-1
. Magic angle spinning-
nuclear magnetic resonance (MAS-NMR) was conducted
on each of the glasses and the resulting spectra are pre-
sented in Fig. 5. Figure 5a presents the spectra of Ly-
Fig. 1 X-ray diffraction and
thermal proﬁle of Ly-N, Ly-C,
Fig. 2 Network connectivity of
glass series calculated (Calc.)
and determined by X-ray
ﬂuorescence (XRF) and
hardness testing of each glass
Table 1 Original glass compositions and (composition determined by
XRF) all in mol. fraction
Ly-N Ly-C Ly-S
SiO2 0.55 (0.53) 0.55 (0.53) 0.55 (0.56)
TiO2 0.05 (0.05) 0.05 (0.05) 0.05 (0.05)
CaO 0.22 (0.23) 0.22 (0.23) 0.22 (0.22)
Na2O 0.18 (0.18) 0.09 (0.09) 0.00 (0.00)
SrO 0.00 (0.00) 0.09 (0.09) 0.18 (0.17)
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N which exhibited a peak at -84.1 ppm. De-convolution of
the peak resulted in a large peak present at -81.8 ppm with
a smaller peak present at -90.1 ppm. An additional peak
can be identiﬁed at -102.1 ppm. Figure 5b shows the
spectrum for Ly-C which produced a peak that was slightly
shifted in the negative direction and centered at
-84.8 ppm. Peak resolution also revealed three peak
positions which are also shifted in the negative direction to
-83.1, -91.4 and -103.1 ppm respectively. Ly-S (Fig. 5c)
experienced a shift further in the negative direction to
-85.1 ppm. The three resolved peaks are centered at
-83.8, -92.6 and -103.2 ppm respectively.
3.2 Investigating glass solubility
Ion release studies were conducted to determine if any
signiﬁcant changes in ion release occurs as the incubation
media is exchanged after 1, 7, 14 and 21 days. To inves-
tigate the solubility of these glasses as a function of Na?
incorporation, particle characterization was performed
prior to ion release studies. Particle size analysis revealed a
similar size distribution for each glass (Table 3) which
were 3.9 lm (Ly-S), 4.7 lm (Ly-C) and 4.6 lm (Ly-
N).Additionally, surface area analysis (Table 3) presented
similar values at 0.97 m2
/g (Ly-S), 0.89 m2
/g (Ly-C) and
/g (Ly-N). Ion release studies were conducted on
each glass at 1, 7, 14 and 21 days with exchange of ﬂuids at
each time period. Regarding Ly-N (Fig. 6a), Si4?
was initially 852 mg/L (1 day), increased to 1107 mg/L
(7 day) and reduced to 664 and 633 mg/L at 14 and
21 days respectively. Na?
release from Ly-N presented a
consistent reduction in release from 1,006 mg/L (1 day),
reduced to 819 mg/L (7 day) and then further to 484 and
451 mg/L at 14 and 21 days respectively.Ca2?
much lower than Si4?
and was consistent over
time where it was 9 mg/L (1 day), 8 mg/L (7 day) and 9
and 10 mg/L at 14 and 21 days respectively. Ion release
from Ly-C considered Si4?
presented in Fig. 6b. Si4?
release was much lower than Ly-
N at 241 mg/L (1 day), increased to 298 mg/L at 7 days
and reduced to 253 and 279 mg/L at 14 and 21 days
release from Ly-C initially presented
Fig. 3 XPS high resolution O
1 s scan of Ly-N, Ly-C, Ly-S
Table 2 High resolution XPS data (Binding Energy, eV)
Si 2p Ca 2p Ti 2p Na 1s Sr 3d
Ly-S 102.1 346.9 458.6 – 133.5
Ly-C 101.8 346.6 458.4 1,071.2 133.2
Ly-N 101.5 346.4 458.2 1,070.6 –
Fig. 4 Raman spectroscopy of, a Ly-N, b Ly-C and c Ly-S
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similar values to Si4?
at 205 mg/L (1 day), but reduced to
134 mg/L (7 day) and then further reduced to 95 and
95 mg/L at 14 and 21 days respectively, a trend similar to
proﬁle of Ly-N. Ca2?
release was also much lower
and ranged from 5 mg/L (1 day),
18 mg/L (7 day), 20 mg/L (14 day) and 6 mg/L (21 day).
release was similar to that of Ca2?
where it ranged
from 3 mg/L (1 day), 20 mg/L (7 day), 27 mg/L (14 day)
and 2 mg/L (21 day). The ion release proﬁle for Ly-S con-
and is presented in Fig. 6c. Si4?
release was found to be consistent over time, similar to Ly-
C at 211 mg/L (1 day), 225 mg/L (7 day), 221 mg/L
(7 day) and 220 mg/L (14 day). Ca2?
release was again
much lower than Si4?
and was consistent over time where
it ranged from 31 mg/L (1 day), 34 mg/L (7 day) and 27
and 25 mg/L at 14 and 21 days respectively. Sr2?
was higher than Ca2?
where it ranged from 62 mg/L
(1 day), 94 mg/L (7 day), 65 mg/L (14 day) and 53 mg/L
(21 day). pH values were recorded at each time period for
each glass and are presented in Fig. 7. Considering Ly-N,
there were relatively minor changes where the pH changed
from 10.6 to 11.3 over 1–14 days and reduced to 10.7 after
21 days. Ly-C experienced a similar trend, however, lower
pH values were recorded at 10.3–10.6 over 1–14 days and
9.8 after 21 days. Similarly, pH values attributed to Ly-
S experienced lower pH values than Ly-C at 10.1–10.4 over
1–14 days and reduced to 9.8 after 21 days.
The effect of Na?
incorporation into the glass
on living cells was investigated using MC3T3 Osteoblasts
to determine if cell adhesion and cell viability was sig-
niﬁcantly inﬂuenced. Cell viability results are presented in
Fig. 8 and revealed that Ly-N presented an insigniﬁcant
change compared to the control cell population after 24 h
(97 %) and 48 h (90 %). Ly-C showed a slight increase at
24 h (107 %) and increased further after 48 h (122 %) and
regarding Ly-S, cell viability increased after 24 h (122 %)
but decreased after 48 h (91 %). Cell adhesion was also
monitored osver 24–48 h and SEM images are presented in
Fig. 9. For each glass, Ly-N, Ly-C and Ly-S, there was
osteoblast attachment after both 24 and 48 h incubation.
4.1 Glass characterization
This study was conducted to determine the effect of Na?
on the structure and dissolution of bioactive
glasses (Table 4). XRD revealed each starting material to
be amorphous while DTA determined an increase in the Tg
from 591 to 760 °C with the substitution of Sr2?
an observable trend is that the Tg increases; Ly-N Ly-
C Ly-S. Further characterization conﬁrmed the glass
composition and determined the speciﬁc role that Na?
play within the glass structure. Both of these ions are
known to perform a similar role in a glass where they act as
network modiﬁers which cause de-polymerization of the
Si–O–Si bonds resulting in the formation of NBO-
Fig. 5 MAS-NMR spectra of
a Ly-N, b Ly-C and c Ly-S
Table 3 Particle size and surface area of each glass powder
Particle size (lm) (S.D.) Surface area (m2
Ly-S 3.9 (0.14) 0.97 (0.07)
Ly-C 4.7 (0.38) 0.89 (0.06)
Ly-N 4.6 (0.15) 1.02 (0.10)
85 Page 8 of 12 J Mater Sci: Mater Med (2015) 26:85
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. Conﬁrming Na?
precise role in the glass will
eliminate differences in solubility based on any glass
structure differences. Initially, network connectivity (NC)
calculations were used to theoretically predict the
speciation within the glass. For this
study NC were used to predict the glass structure assuming
TiO2 acts as a network modiﬁer and also as a network
former and compared to experimental data collected using
x-ray ﬂuorescence. The calculated and predicted NC was
determined to be very similar. In order to validate the NC
predictions, complementary techniques were used for
evaluating glass structure including, high resolution XPS,
Raman Spectroscopy and MAS-NMR. With respect to high
resolution XPS, deconvolution of the BO signal
(531.3–531.8 eV) and the NBO-
revealed a slight shift in BE, which was observed at higher
BE as Sr2?
is increased within the glass, however, the ratio
of BO:NBO was similar for each material at 45:55. An
established method of representing the atomic structural
arrangement or network connectivity of a glass in terms of
structural units can be represented by Qn
units, where Q
represents the Si tetrahedral unit and n the number of
bridging oxygens (BO); where n ranges between 0 and 4.
is the central tetrahedral atom which ranges from Q0
(orthosilicates) to Q4
(tectosilicates) and Q1
structures representing intermediate silicates containing
modifying oxides . Determining the Q-structure of the
glass yields structural information about the local envi-
ronment around the Si atom which can be determined using
Raman spectroscopy and MAS-NMR. Considering Raman
data, it is also evident that there is a slight shift in the
spectral envelope towards higher wavenumbers in the
Fig. 6 Ion release of a Ly-N, b Ly-C and c Ly-S over 1, 7, 14 and
Fig. 7 pH of Ly-N, Ly-C and Ly-S over 1, 7, 14 and 21 days
Fig. 8 Cell viability of Ly-N, Ly-C and Ly-S after 24 and 48 h in MC
J Mater Sci: Mater Med (2015) 26:85 Page 9 of 12 85
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region of 850–875 cm-1
Millan et al. assigns the
wavenumbers representing Q4
to 1060–1,200, Q3
to 1,000–950, Q1
to 900 and Q0
. The Raman data within this region present relatively
similar peaks at 850–870 cm-1
, which is indicative of a
highly disrupted glass network, 4NBO/Si. However, the
broadening of the spectral envelope to higher wavenum-
bers, particularly with respect to Ly-S, is indicative
increasing BO content. Additional bands located in the
region of 600 cm-1
have previously been described by
Fig. 9 Cell adhesion of a Ly-N,
b Ly-C and c Ly-S over 24 and
48 h using MC-3T3 Osteoblasts
Table 4 Summary of glass
structure and characterization
Net. conn. XPS (BE) Raman (cm-1
) MAS-NMR (ppm) Q-structure
Theo. XRF BO NBO
Ly-S 2.36 2.26 531.8 530.1 852 -85.1 Q1/Q2
Ly-C 2.36 2.26 531.6 529.9 861 -84.8 Q1/Q2
Ly-N 2.36 2.42 531.3 529.7 873 -84.1 Q1/Q2
85 Page 10 of 12 J Mater Sci: Mater Med (2015) 26:85
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Aguiar et al. as being related to ring structures, with this
speciﬁc region being related to three-membered rings .
MAS-NMR data corroborates high resolution XPS and
Raman spectroscopy where the peak positions for each
material are similar at -84 ppm (Ly-N, Ly-C) and
-85 ppm (Ly-S). Previous NMR studies by Galliano et al.
and Hayakawa et al. on silicate melts suggest the presence
species at -78, -85 and -95 ppm
respectively [17, 18]. With respect to the NMR shift evi-
dent in this study, the Ly-N produced resolved peaks at
lower ppm than Ly-C, with Ly-S presenting ppm shifts in a
more negative direction in each case. This suggests that
each glass contains a distribution of Q-species, predomi-
. This is a positive attribute as dissolution ion
exchange from bioactive glass is favored by a high con-
centration of NBO-
species and low Q-speciation .
Glass characterization determined very little difference in
the glass structure in relation to the glass network con-
nectivity and BO/NBO content, however, DTA and hard-
ness testing suggest that the incorporation of Sr2?
encourages more resilient bonds within the glass network
as evinced by the increase in Tg (Ly-N 591 °C, Ly-
S 760 °C) and the signiﬁcant increase in hardness with
incorporation, as the hardness associated with Ly-
N was signiﬁcantly lower than Ly-C (P = 0.000) and Ly-
S (P = 0.000), however no signiﬁcant difference exists
between Ly-C and Ly-S (P = 0.852). This shift in Tg and
the increase in hardness may be due to the fact that
can charge compensate a single NBO-
while a single divalent Sr2?
ion can charge compensate
. This essentially results in two fold increase in
charge compensated Si-NBO-
species within the glass
with the addition of Sr2? (Scheme 1).
4.2 Investigating glass solubility
Particle size analysis and surface area analysis proved that
there were no signiﬁcant differences in particle size/surface
area that would contribute to difference in ion release data.
Additionally, glass characterization determined that the
relative concentration of BO to NBO was similar for each
glass, hence the dissolution of the glass should be based on
the characteristics of the ions (Na?
) present and not
related to signiﬁcant differences in the glass structure or
particle effects. The Na?
release proﬁles demonstrated here
are higher for Ly-N compared to BioglassÒ
, (190–270 mg/
L after 30 days) however, Ly-C and Ly-S are comparable
. Regarding Ly-N, the highest ion release rates were
recorded for Na?
which was found to reduce with each
ﬂuid exchange up to 21 days. A similar trend was present
with Ly-C which suggests that Na?
depletion from the
glass particles is occurring. Si4?
release from bioactive
glass is essential for the formation and calciﬁcation of bone
tissue and is known to increase bone mineral density.
is also known to induce Hap precipitation
and Si(OH)4 stimulates collagen I formation and osteo-
blastic differentiation . Si4?
release from BioglassÒ
determined levels much lower than reported here at 5 mg/L
after 1 day, 20 mg/L at 7 days and 45 mg/L after 30 days
release was greatly reduced with the addition of
to the glass, Ly-N (800–1,100 mg/L), whereas Ly-C,
Ly-S, (200–300 mg/L). This is likely due to Sr2?
a more stable bond between NBO-
groups within the glass
which essentially forms cross-bridges that stabilize the
tetrahedron. This is also supported by the differences
in Tg and hardness between Ly-N and Ly-C/Ly-S. Ca2?
release from bioactive glass is known to promote dissolu-
tion of the glass surface. This characteristic is essential for
encouraging precipitation of a calcium phosphate surface
layer in vivo . Ca2?
is also cited to encourage osteo-
blast proliferation, differentiation and extracellular (ECM)
mineralization [6, 20]. Regarding this study, Ca2?
proved to be relatively consistent within each glass, and did
not decrease even with ﬂuid exchange at each time period.
This suggests that Ca2?
may be reaching a solubility limit
which ranges from 9 to 10 mg/L (Ly-N), 5–20 mg/L (Ly-
C) and 25-34 mg/L (Ly-S). Ca2?
release from BioglassÒ
are within the approximate levels cited here at 7.5 mg/L
(1 day), 10 mg/L (7 days) to 16 mg/L (30 days) . Sr2?
release ranged from 2 to 27 mg/L in Ly-C, however, it
increased to 53–94 mg/L in Ly-S which is likely due to the
increase in Sr2?
concentration in the glass. An associated
inﬂuence of the glass solubility in addition to ion release is
changes in solution pH. The solution pH was found to
decrease as the Na?
concentration in the glasses is reduced
and/or eliminated. The lowest pH values were recorded
with Ly-C and Ly-S after 21 days. In the case of Ly-C this
Scheme 1 a Sodium and b strontium charge compensating NBO-
species in a silicate glass network
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effect is likely due to the reduction in Na?
with regard to Ly-S the likely reason is that the Sr2?
are lowest at 21 days at 53 mg/L. The biocompatibility of
each glass was evaluated using cytotoxicity analysis and
cell adhesion studies. Cytotoxicity analysis at 24 h deter-
mined that there was no signiﬁcant difference in cell via-
bility when comparing the growing cell population to Ly-
N (P = 1.000) or Ly-C (P = 1.000), however, Ly-S pre-
sented a signiﬁcant increase in cell viability (P = 0.012).
Regarding the 48 h samples, there was no signiﬁcant dif-
ference between the growing cell population and Ly-
N (P = 1.000), Ly-C (P = 1.000) and Ly-S (P = 1.000).
To further support the lack of cytotoxicity, cell adhesion
studies determined that each composition studied sup-
ported the adherence of osteoblast cells to the materials
surface. The cell culture data determined that the materials
under evaluation did not prove toxic to osteoblast cells
after 24 or 48 h despite the difference in glass solubility.
within this glass system resulted
in insigniﬁcant changes in glass structure as determined by
XPS, Raman Spectroscopy and MAS-NMR, however, the
addition of Sr2?
greatly increased bond strength within the
glass resulting in a higher Tg and hardness values. The
additions of Sr2?
also greatly reduced the solubility of the
glass and reduced the solution pH, however, there were no
signiﬁcant difference in cell viability and adhesion asso-
ciated with the difference in glass solubility. Future work
will aim to look at how the difference in glass solubility
inﬂuences precipitation of a calcium phosphate layer in
simulated body ﬂuid on glass plates, and to quantitatively
determining preference for cell adhesion on solid glass
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