sodium and strontium -structure and solubility

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Journal of Materials Science:Materials in MedicineOfficial Journal of the European Societyfor Biomaterials ISSN 0957-4530Volume 26Number 2 J Mater Sci: Mater Med (2015) 26:1-12DOI 10.1007/s10856-015-5415-5

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

1 23

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BIOMATERIALS SYNTHESIS AND CHARACTERIZATION

Investigating the influence of Na+ and Sr2+ on the structureand 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

� Springer Science+Business Media New York 2015

Abstract This study was conducted to determine the

influence that network modifiers, sodium (Na?) and stron-

tium (Sr2?), 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

Sr2? containing glasses. Additionally the Sr2? (*250 mg/L)

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.

1 Introduction

Bioactive glasses have generated considerable interest in the

recent past as a medical material. Since the inception of

Bioglass� in the late 1960s by Prof Larry Hench, numerous

glass compositions have been investigated for their thera-

peutic potential [1]. The original composition (45S5 Bio-

glass�), 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

[4]. 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 Bioglass� 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

[6]. In particular, these reactions involve dissolution of criti-

cal concentrations of soluble Si4? and Ca2? ions that gives

rise to both intracellular and extracellular responses at the

interface of the glass with its physiological environment [4].

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

(Ca2?, Na?) from the glass react with H? (H3O) ions from the

solution leads to hydrolysis of the silica groups with the

Y. Li � L. M. Placek � D. Pradhan � N. P. Mellott �A. W. Wren (&)

Inamori School of Engineering, Alfred University, Alfred,

NY 14802, USA

e-mail: [email protected]

A. Coughlan

School of Materials Engineering, Purdue University,

West Lafayette, IN, USA

F. R. Laffir

Materials and Surface Science Institute, University of Limerick,

Limerick, Ireland

123

J Mater Sci: Mater Med (2015) 26:85

DOI 10.1007/s10856-015-5415-5

Author's personal copy

creation of Silanol (Si–OH) [1, 4, 7]. Condensation of an

amorphous Si-rich layer (depleted in Ca2? and Na?), pro-

ceeds on the glass surface followed by migration of Ca2? and

PO43- 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

OH- and CO32- 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? and Ca2? in

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

(Na?, K?, Ca2?) 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

(Si–O–NBO-). 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

monovalent (Na?) and a divalent (Sr2?) cation can have on the

structure of a bioactive glass, and the subsequent solubility.

Na? was selected as the monovalent cation as its role in bio-

active glasses has been well described [4, 7], and Sr2? is

known to have positive therapeutic effects in vivo, where it has

been cited as increasing the proliferation of osteoblasts while

reducing osteoclastic activity [12]. 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?, Sr2?) act

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? and Sr2? addition. The

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

Strontium (Sr2?, Ly-S) incorporation. A control glass (Ly-

C) was also formulated which contained equal quantities of

Na? and Sr2?. 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


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


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

85 Page 2 of 12 J Mater Sci: Mater Med (2015) 26:85

123


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.

NC ¼ No:BOs� No:NBOs

Total No:Bridging Speciesð1Þ

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

(MAS-NMR)

29SiMAS 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. 29SiMAS

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. 29Si 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-

line correction.

J Mater Sci: Mater Med (2015) 26:85 Page 3 of 12 85

123


2.3 Investigating glass solubility

2.3.1 Particle size analysis (PSA)

Particle size analysis was conducted using a Beckman Coulter

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?),

Silicon (Si4?), Titanium (Ti4?), Calcium (Ca2?) and

Strontium (Sr2?) 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


123


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

Spectrometer.

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 Results


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.

A Na? containing glass (Ly-N), an intermediate glass

containing both Na? and Sr2? (Ly-C), and a Sr2? con-

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? concentration

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?/Sr2? replacement. High

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

Sr2? and Na? 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? containing

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


123


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. A

slight shift in wavenumbers was observed within the region

of 800–1,000 cm-1. Ly-N (Fig. 4a) presented a peak at

873 cm-1 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

861 cm-1which 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,

Ly-S

Fig. 2 Network connectivity of

glass series calculated (Calc.)

and determined by X-ray

fluorescence (XRF) and

hardness testing of each glass

surface

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)


123


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.


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?/

Sr2? 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

1.02 m2/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? release

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? release was

much lower than Si4? and Na? 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?, Na?, Ca2? and Sr2? and is

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

respectively. Na? 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


123


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

the Na? profile of Ly-N. Ca2? release was also much lower

than Si4? and Na? and ranged from 5 mg/L (1 day),

18 mg/L (7 day), 20 mg/L (14 day) and 6 mg/L (21 day).

Sr2? 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-

siders Si4?, Ca2? and Sr2? 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? release

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? and Sr2? 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 Discussion


This study was conducted to determine the effect of Na?

and Sr2? 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? for Na?,

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? and

Sr2? 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- species

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/g) (S.D.)

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)


123


[6]. Confirming Na? and Sr2? 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

approximate NBO-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- (529.7–530.1 eV)

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.

Si4? is the central tetrahedral atom which ranges from Q0

(orthosilicates) to Q4 (tectosilicates) and Q1, Q2 and Q3

structures representing intermediate silicates containing

modifying oxides [15]. 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

21 days

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

3T3 Osteoblasts


123


region of 850–875 cm-1. McMillan et al. assigns the

wavenumbers representing Q4to 1060–1,200, Q3 to

1,100–1,050, Q2 to 1,000–950, Q1 to 900 and Q0 to 850

[16]. 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 characterizationNet. 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


123


Aguiar et al. as being related to ring structures, with this

specific region being related to three-membered rings [15].

Si29MAS-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

of Q1, Q2 and Q3 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-

nantly Q1/Q2. 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 [6].

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

Sr2? 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

monovalent Na? can charge compensate a single NBO-,

while a single divalent Sr2? ion can charge compensate

2NBO-. This essentially results in two fold increase in

charge compensated Si-NBO- species within the glass

with the addition of Sr2? (Scheme 1).


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?, Sr2?) 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 Bioglass�, (190–270 mg/

L after 30 days) however, Ly-C and Ly-S are comparable

[19]. 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.

Aqueous Si4? is also known to induce Hap precipitation

and Si(OH)4 stimulates collagen I formation and osteo-

blastic differentiation [20]. Si4? release from Bioglass�

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

[19]. Si4? release was greatly reduced with the addition of

Sr2? 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? providing

a more stable bond between NBO- groups within the glass

which essentially forms cross-bridges that stabilize the

Si4? 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 [21]. Ca2? is also cited to encourage osteo-

blast proliferation, differentiation and extracellular (ECM)

mineralization [6, 20]. Regarding this study, Ca2? release

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 Bioglass�

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) [19]. 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 thisScheme 1 a Sodium and b strontium charge compensating NBO-

species in a silicate glass network


123


effect is likely due to the reduction in Na? release, and

with regard to Ly-S the likely reason is that the Sr2? levels

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.

5 Conclusion

Substituting Na? and Sr2? 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

samples.

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sodium and strontium -structure and solubility

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