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Full length article
In vitro osteogenesis by intracellular uptake of strontium containing
bioactive glass nanoparticles
Parichart Naruphontjirakul, Alexandra E. Porter, Julian R. Jones
⇑
Department of Materials, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
article info
Article history:
Received 11 August 2017
Received in revised form 5 October 2017
Accepted 7 November 2017
Available online 10 November 2017
Keywords:
Bioactive glass nanoparticles
Osteogenic
Strontium
Intracellular uptake
Endocytosis
abstract
Monodispersed strontium containing bioactive glass nanoparticles (Sr-BGNPs) with two compositions
were synthesised, through a modified sol-gel Stöber process, wherein silica nanoparticles (SiO
2
-NPs)
were formed prior to incorporation of calcium and strontium, with diameters of 90 ± 10 nm. The osteo-
genic response of a murine preosteoblast cell line, MC3T3-E1, was investigated in vitro for a nanoparticle
concentration of 250 mg/mL with compositions of 87 mol% SiO
2
, 7 mol% CaO, 6 mol% SrO and 83 mol%
SiO
2
, 3 mol% CaO, 14 mol% SrO. Dissolution studies in minimum essential media (
a
-MEM) at pH 7.4
and artificial lysosomal fluid (ALF) at pH 4.5 showed that the particles dissolved and that Sr
2+
ions were
released from Sr-BGNPs in both environments. Both particle compositions and their ionic dissolution
products enhanced the alkaline phosphatase (ALP) activity of the cells and calcium deposition.
Immunohistochemistry (IHC) staining of Col1a1, osteocalcin (OSC) and osteopontin (OSP) showed that
these proteins were expressed in the MC3T3-E1 cells following three weeks of culture. In the basal con-
dition, the late osteogenic differentiation markers, OSC and OSP, were more overtly expressed by cells
cultured with Sr-BGNPs with 14 mol% SrO and their ionic release products than in the control condition.
Col1a1 expression was only slightly enhanced in the basal condition, but was enhanced further by the
osteogenic supplements. These data demonstrate that Sr-BGNPs accelerate mineralisation without osteo-
genic supplements. Sr-BGNPs were internalised into MC3T3-E1 cells by endocytosis and stimulated
osteogenic differentiation of the pre-osteoblast cell line. Sr-BGNPs are likely to be beneficial for bone
regeneration and the observed osteogenic effects of these particles can be attributed to their ionic release
products.
Statement of Significance
We report, for the first time, that monodispersed bioactive glass nanoparticles (90 nm) are internalised
into preosteoblast cells by endocytosis but by unspecific mechanisms. The bioactive nanoparticles and
their dissolution products (without the particles present) stimulated the expression of osteogenic mark-
ers from preosteoblast cells without the addition of other osteogenic supplements.
Incorporating Sr into the bioactive glass nanoparticle composition, in addition to Ca, increased the total
cation content (and therefore dissolution rate) of the nanoparticles, even though nominal total cation
addition was constant, without changing size or morphology.
Increasing Sr content in the nanoparticles and in their dissolution products enhanced osteogenesis
in vitro. The particles therefore have great potential as an injectable therapeutic for bone regeneration,
particularly in patients with osteoporosis, for which Sr is known to be therapeutic agent.
Ó2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Bioactive glass-based products are used for several orthopaedic
and dental applications [1] because they form strong bonds with
host bone [1–4] and the ions released from these products stimu-
late osteogenic gene expression, leading to rapid bone regeneration
[5–9]. Recently, the development of new formulations by introduc-
tion of other therapeutic metallic cations, such as copper, zinc,
cobalt into the silica network, and using co-networks of borate,
have widened the potential therapeutic applications to angiogene-
sis, wound healing, antimicrobial and anti-cancer [9–12].
https://doi.org/10.1016/j.actbio.2017.11.008
1742-7061/Ó2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
⇑
Corresponding author.
E-mail address: julian.r.jones@imperial.ac.uk (J.R. Jones).
Acta Biomaterialia 66 (2018) 67–80
Contents lists available at ScienceDirect
Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat
Strontium (Sr) has been clinically used as pharmaceutical agent for
osteoporosis treatment [13,14] because of its ability to activate
osteoblasts and inhibit osteoclast activities [15–18]. Strontium
ions (Sr
2+
) have a similar charge and ionic radius to calcium ions
(Ca
2+
)[15]. SrO can therefore replace some CaO in the bioactive
glass structure [15,19–21]. Sr is slightly larger and heavier com-
pared with Ca, therefore the substitution decreases the connectiv-
ity of the silica network by further disrupting the network [11,22].
Bioactive glass nanoparticles (BGNPs) are promising injectable
biomaterials for bone regeneration applications [12,23,24]. The
potential benefits of nanoparticles (NPs) over microparticles are
their high surface to volume ratios, which increases their dissolu-
tion rate [17,25,26], and their small size allows them to be inter-
nalised into cells to deliver their therapeutic ions intracellularly
[26,27]. The benefit of the glass is that it can provide a sustained
delivery of the therapeutic cations from their amorphous structure
during dissolution [6,16,26,28].
The size and shape of NPs is known to affect the endocytosis
pathway [29–33]. NPs internalise and localise within the cells
through different uptake pathways, including phagocytosis,
macropinocytosis, clathrin-independent endocytosis, and
clathrin-dependent endocytosis [34,35]. To explore the potential
utility of Sr-BGNPs as therapeutic cation carriers, it is critical to
understand the different mechanisms of Sr-BGNPs uptake by cells.
SiO
2
-CaO NPs with diameters of 215 ± 20 nm were previously
found to be internalised into human bone marrow and adipose
derived stem cells and internalization by the stem cells was not
affected following inhibition of clathrin- or caveolin- mediated
endocytosis [35].
A previous study [36] showed that cation incorporation into the
silica network was not trivial, indicating that previous studies had
overestimated the amount of calcium incorporated into nanoparti-
cles, without checking composition. The most effective method to
incorporate calcium ions into the NPs is by adding calcium nitrate,
after the monodispersed SiO
2
-NPs has already been formed [36].
This modified Stöber method was adapted to produce SiO
2
-CaO-
SrO NPs [37]. In each case, not all of the nominal Ca
2+
and Sr
2+
ions
were incorporated into the silica network [36,37]. There is an
upper limit of network modifier incorporation into the dense
SiO
2
-NPs. A maximum of 10% mol CaO incorporation into the bin-
ary BGNPs (90% mol SiO
2
and 10% mol CaO), using a nominal ratio
of Si:Ca of 1:1.3, was possible using a modified Stöber process [36].
The upper limit of Sr
2+
ion incorporation into the binary BGNPs was
approximately 17 mol% (83% mol SiO
2
and 17% mol SrO), using the
nominal ratio of Si:Sr of 1:1.3 [37]. Substituting Sr
2+
for Ca
2+
ions
on a molar basis had no effect on the size and morphology of the
particles. The in vitro cell viability also showed that the Sr-BGNPs
had low cytotoxicity (>70% viability) for particle concentrations
of up to 250
l
g/mL and the ions released from these particles sig-
nificantly increased the viability of the MC3T3-E1 osteoblast-like
cells at concentrations of 200 and 250
l
g/mL [37]. Only cell viabil-
ity was assessed.
Here, the aim was to investigate the effect of Sr-BGNPs on the
osteogenic response of MC3T3-E1 cells in vitro using Sr-BGNPs,
with diameters within the range of 80–100 nm, and the influ-
ence of mol% of SrO. The mechanism of incorporation of the
nanoparticles into the cells was also investigated. To understand
whether or not the therapeutic effect is dependent on intracellu-
lar release of therapeutic ions from the particles, the cells were
exposed to both the particles and their dissolution products.
Ion release from the Sr-BGNPs was also measured in three differ-
ent conditions: phosphate-buffered saline (PBS), minimum
essential media (
a
-MEM) at pH 7.4 and artificial lysosomal fluid
(ALF) at pH 4.5.
2. Materials and methods
2.1. Sr-BGNPs synthesis
All reagents were from Sigma–Aldrich (Dorset, UK) unless sta-
ted otherwise. SiO
2
-NPs and Sr-BGNPs, with diameters of 90
nm, were prepared using the modified Stöber method described
previously [36,37]. A diameter of 90 nm was chosen because of
previous work on size dependence on internalisation of Stöber-
like particles, where particles of <200 nm were internalised into
cells [38,39] and particles of 60 nm caused higher toxicity than
100 nm particles [40], which did not cause toxicity up to the con-
centration of 500 mg/mL [41].
For their synthesis, first, 32.92 mL of ethanol (99.5%), 4.11 mL of
distilled water, and 0.48 mL of ammonium hydroxide were mixed
in an ultrasonication bath for 10 min. Then, 2.50 mL of tetraethyl
orthosilicate (TEOS) was gently added to the mixed solution and
left in the ultrasonication bath at least 6 h to complete hydrolysis
and polycondensation reactions [37]. Hydrolysis and condensation
reactions of TEOS occurred simultaneously to form the silica net-
work (Si-O-Si). SiO
2
-NPs were centrifuged for collection and then
were washed with ethanol (two times) and distilled water.
For Ca and Sr incorporation, calcium nitrate tetrahydrate (99%)
and strontium nitrate tetrahydrate (99%) were added. Based on
previous work [37], a nominal molar ratio of 1:1.3 ratio of Si:total
cations was selected. Binary BGNPs (SiO
2
-CaO) and ternary BGNPs
(SiO
2
-CaO-SrO) compositions were synthesised, with 25 mol% and
75 mol% calcium nitrate tetrahydrate being replaced with stron-
tium nitrate tetrahydrate, leaving SiO
2
-CaO-SrO compositions con-
taining 6.2 mol% SrO (6%Sr-BGNPs) and 14.2 mol% SrO (14%Sr-
BGNPs) respectively (measured by acid digestion, Section 2.2).
The resulting primary particle suspension was then dried at 60 °C
overnight to remove excess water following with thermal treat-
ment at 680 °C for 3 h at a heating rate 3 °C/min in order to pro-
duce Sr-BGNPs. These particles were then washed with ethanol
two times.
2.2. Acid digestion compositional analysis
Acid digestion compositional analysis was carried out to mea-
sure the composition of the BGNPs by the lithium metaborate
fusion dissolution method. 50 mg of finely ground particles was
mixed carefully with 250 mg of anhydrous lithium metaborate
(80% w/w) and lithium tetraborate (20% w/w) (Spectroflux 100B,
Alfa Aesar, Lancashire, UK) in a clean and dry platinum crucible
using a glass rod [42]. The mixture was fused in a furnace for
20 min at 1050 °C and later dropped to room temperature. The
mixture was subsequently dissolved in 2 M nitric acid [43].
The elemental concentration in the solution was measured using
inductively coupled plasma optical emission spectroscopy (ICP-
OES, Thermo Scientific iCAP 6000 series).
2.3. Particle characterization
Particle size was investigated using Dynamic Light Scattering
(DLS, Malvern instrument 2000) and Transmission Electron Micro-
scopy (TEM, JEOL 2100 Plus microscope operated at 200 kV). To
prepare samples, the dried particles were dissolved in ethanol
and sonicated in the sonication bath for 15 min before conducting
the DLS measurements. Particles were collected on 400 mesh cop-
per transmission electron microscopy (TEM) grids, coated with
holey carbon film (TAAB, Berkshire, UK). TEM images were used
to confirm particles’ size and morphology.
68 P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80
To indicate the stability of particles in solutions, Zeta (f) poten-
tial values were measured in distilled water in three different pH:
3.0, 7.4, and 11.0 using Zeta sizer (Malvern instrument 2000).
X-ray Diffraction (XRD) patterns were collected with a Philips
PW1700 series automated powder diffractometer using Cu K
a
radiation (1.54 Å) at 40 kV/40 mA. Data was collected in the 10–
70°2hrange with a step size of 0.04°and a dwell time of 1.0 s to
identify the crystallised pattern of the particles.
2.4. Dissolution study
To compare the release rate of ions from the Sr-BGNPs with dif-
ferent mol% SrO, the release of Si, Ca and Sr ions from Sr-BGNPs
was evaluated as a function of time in three different solutions:
minimum essential media (
a
-MEM) medium (Thermo Fisher Sci-
entific, Hemel Hempstead, UK) at pH 7.4, artificial lysosomal fluid
(ALF) at pH 4.5 (Supplementary information Table S1) and
phosphate-buffered saline (PBS) at pH 7.4. 75 mg of BGNPs were
suspended with 5 mL of media in dialysis tubing, that had a molec-
ular weight cut-off of 10 kDa, and immersed into 45 mL of media
[42]. All samples were incubated at 37 °C with continuous shaking
at 120 rpm for 1, 2, 3, 4, 5, 6, 7, 24, 48, 72, 96, 168, 240 h in PBS and
ALF and for 1, 2, 3, 4, 5, 6, 7, 24 h in
a
-MEM medium. At each of the
time intervals, 1 mL of the bulk solution was collected and then
immediately replaced with 1 mL of the fresh solution. The pH of
solution was monitored at each specific interval over a period of
240 h in PBS and ALF and for 24 h in
a
-MEM.
The collected solution was diluted in distilled water (for the
a
-
MEM medium) and 2 M nitric acid (for ALF and PBS) with a 10-fold
dilution factor. The elemental concentrations of Si, Ca, and Sr were
measured using ICP-OES (Thermo Scientific iCAP 6000 series).
At the end of incubation period, the Sr-BGNPs were washed
with ethanol and acetone to terminate any reactions [42] and then
collected on 400 mesh copper TEM grids, coated with a holey car-
bon support film and bright field TEM imaging was conducted.
2.5. In vitro cytotoxicity assay
The next step was to evaluate whether the therapeutic effect of
the Sr-BGNPs could be linked directly to an ionic effect of the Sr
and Ca ions which are released from the particles. A murine pre-
osteoblast cell line, MC3T3-E1 cells (ATCC) was incubated with
both the particles and media containing only their dissolution
products. MC3T3-E1 cells were routinely cultured under standard
condition in a humidified atmosphere at 37 °C and 5% CO
2
in basal
a
-MEM media. These media were supplemented with 10% fetal
bovine serum (FBS) (v/v), 100 U/mL penicillin and 100
l
g/mL
streptomycin (Thermo Fisher Scientific, Hemel Hempstead, UK).
Cells were seeded in flat-bottomed 96-well plates, at a seeding
density of 5 10
4
cells/mL, and incubated at 37 °C and 5% CO
2
for 24 h to allow cells to attach in a monolayer.
Based on our previous report [37], 6%Sr-BGNPs and 14%Sr-
BGNPs did not cause toxicity to the MC3T3-E1 cells up to 250 mg/
mL. Here, effects of the of 6%Sr-BGNPs and 14%Sr-BGNPs on cell
viability was investigated with the extended NP concentration
range from 0 to 1000 mg/mL (0.01, 0.1, 1, 10, 100, 150, 200, 250,
500, 1000) using a pulse-chase exposure, where cells were exposed
to the pulse phase for 24 h, followed by chase period of 1, 3 and 7
days. The effect of the ions released from the 6%Sr-BGNPs and 14%
Sr-BGNPs on cell viability was measured after cells were incubated
with the media retrieved from the dissolution experiments for 1, 3,
and 7 days.
Cell viability was determined using the MTT colorimetric assay
(Thermo Fisher Scientific, Hemel Hempstead, UK) based on the
conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo
lium bromide (MTT) into formazan. The formazan is soluble in
dimethyl sulfoxide (DMSO) and the concentration of soluble for-
mazan was determined at 570 nm using a microplate reader (Spec-
traMax M2
e
, Molecular device).
2.6. Effect of Sr-BGNPs and their ionic release products on MC3T3-E1
differentiation
MC3T3-E1 cells were cultured, using a cell density at 5 10
4
cells/mL, in a flat-bottomed 24-well plate and incubated at 37 °C
and 5% CO
2
for 24 h to allow cells to attach to the plate. Cells were
cultured either in basal
a
-MEM or osteogenic medium (
a
-MEM
supplemented with 100
l
M L-ascorbic acid (Sigma-Aldrich, UK),
10 mM b-glycerophosphate (Sigma-Aldrich, UK) and 10 nM dex-
amethasone (DEX, Sigma-Aldrich, UK)).
Cells were exposed to Sr-BGNPs or the media containing the 6%
Sr-BGNP’s and 14%Sr-BGNP’s ionic release products both in the
basal and osteogenic conditions. Media containing the ionic release
products were made by immersing Sr-BGNPs in the media at con-
centration of 250 mg/mL for 24 h with continuous shaking at 120
rpm. The culture media were changed every three days to ensure
a high nutrient concentration. The NPs were incubated with the
cells with every media change. Cells were fixed with 4%
paraformaldehyde in PBS at time intervals of up to 21 days. Cells
were stained with an alkaline phosphate (ALP) detection kit (Merck
Millipore, Middlesex, UK) according to the manufacturer’s
instructions.
Key osteoblastic differentiations marker staining was carried
out for Collagen type 1 (Col1a1), osteocalcin (OSC), and osteopon-
tin (OSP). After fixation, cells were permeabilised with a perme-
ability buffer for 30 min and then blocked with 1% BSA in PBS for
5 min. The cells were stained with a rabbit IgG primary antibody
(Abcam, Cambridge, UK) at 4 °C overnight. The secondary antibody
used for immunofluorescence was goat anti-rabbit IgG H&L conju-
gated with Alexa Fluor 455 (Abcam, Cambridge, UK). 4
0
,6-Diami
dino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fisher Scien-
tific, Hemel Hempstead, UK) was used to stain nuclei.
Cells were stained with 1% Alizarin Red S in PBS at pH 4.2 to
detect calcified tissue formation.
2.7. Endocytosis study
2.7.1. Fluorescent labelling of nanoparticles
14%Sr-BGNPs were functionalised using a modified method
from previous work [44]. 50 mg of 14%Sr-BGNPs was re-
dispersed in 10 mL absolute ethanol (5% w/v) followed by careful
addition of 500 mL of 28% NH
4
OH. Then, 2 mL of (3-aminopropyl)
triethoxysilane (APTES) was mixed on a shaker at 200 rpm over-
night to complete reactions. Finally, amine functionalised Sr-
BGNPs were completely washed with absolute ethanol two times
to remove the excess components.
After Sr-BGNPs were functionalised with amine groups, the par-
ticles were labelled with fluorescein coupling using a modified
method from previous research [45]. For 1% w/v fluorecein 5(6)-
isothiocyanate bioreage: FITC, 20 mg of FITC was dissolved in abso-
lute ethanol and gently mixed (200 rpm) in dark conditions. Next,
20 mg of amine functionalised Sr-BGNPs was added and stirred for
16 h to complete the reaction. Lastly, FITC-14%Sr-BGNPs were
washed with absolute ethanol (2 times) and D.I. water (once)
(Scheme 1). To investigate the effect of the FITC conjugated BGNPs
(FITC-14%Sr-BGNPs) on the viability of the MCT3T-E1 cells, an MTT
assay was performed.
2.7.2. Endocytosis mechanism inhibition study
To understand the mechanism by which the NPs were inter-
nalised by the cells, MCT3T-E1 cells (cell density at 5 10
4
cells/
mL) were seeded on 6-well plates. After the cells were cultured
P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80 69
under standard conditions, in a humidified atmosphere at 37 °C
and 5% CO
2
in basal
a
-MEM media, the cells were subsequently
incubated with different endocytosis inhibitors for 2 h.
The MC3T3-E1 cells were then pre-treated at 37 °C for 2 h
with five different endocytosis chemical inhibitors including
wortmannin (wor, an inhibitor of phagocytosis); amiloride
hydrochloride hydrate (ami, an inhibitor of macropinocytosis);
chlorpromazine hydrochloride USP (chlor, an inhibitor of
macropinocytosis); genistein (gen, an inhibitor of clathrin-
independent endocytosis) and cytochalasin D (cytD, an inhibitor
of clathrin-dependent endocytosis). The NPs were replaced at
concentration 250 mg/mL and were then incubated for 24 h. The
concentration of endocytosis inhibitors was: 23 mM of wor; 1
mM of ami; 37 mM of gen (VWR International Ltd, Lutterworth,
UK); 4 mM of cytD; and 30 mM of chlor. These concentrations were
selected using previous work as a guide [46]. After 24 h cells were
fixed with 4% paraformaldehyde in PBS.
2.8. Statistics
Statistical analyses were performed by one-way analysis of
variance (ANOVA) in Minitab with the appropriate post hoc com-
parison test (Tukey’s test). A p-value <.05 was considered signifi-
cant. The graphs shown present the results as the mean value
with the standard deviation (SD) as the error bars.
3. Results
3.1. Material characterization
Table 1 shows the compositions of the Sr-BGNPs. Elemental
analysis confirmed that the binary glass had a composition of 93
mol% SiO
2
, 7 mol% CaO. Interestingly, adding a third component
(Sr), while keeping the nominal ratio of Si:total modifying cations
constant, increased the overall amount of network modifiers in the
stabilised NPs (13 mol% and 17 mol% CaO + SrO), compared with
the binary NPs containing only CaO (7 mol% CaO). This increase
might be due to the larger ionic radius of the Sr compared to Ca,
causing the silicate network to be more open, allowing diffusion
of both cations into the NPs during thermal stabilisation. However,
this does not explain the reduction in Ca content.
Scheme 1. (a) Sr-BGNPs conjugation with FITC and (b) indirect measurement of FITC-Sr-BGNPs endocytosis mechanism.
Table 1
Composition of the synthesised Sr-BGNPs (mol%).
Samples Mol%
SiO
2
CaO SrO
0%Sr-BGNPs 92.8 ± 1.3 7.2 ± 0.2 0.0 ± 0.0
6%Sr-BGNPs 87.1 ± 0.3 6.7 ± 0.2 6.2 ± 0.0
14%Sr-BGNPs 83.3 ± 0.2 2.5 ± 0.2 14.2 ± 0.0
70 P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80
The morphology of the batches and particle diameters agreed
with that found previously [37]. The different compositions of
the BGNPs, for the different Ca and Sr contents, did not influence
the size and morphology of the particles. TEM images of dense
monodisperse Sr and Si-BGNPs (Fig. 1) showed that they all had
spherical morphology. Fig. 2 shows their size distribution mea-
sured using DLS with modal diameters of 90 ± 10 nm.
As an indicator of the stability of particles in water, and the
effect of changing pH, the f-potential of the particles of were mea-
sured by suspending them in DI water at three different pH values:
3.0, 7.4 and 11. (Table 2). The f-potential values ranged from 1.5 ±
0.1 to 4.6 ± 0.3 mV (pH 3.0), 22.6 ± 0.6 to 29.7 ± 1.3 mV (p7.4)
and 32.6 ± 1.6 to 41.6 ± 1.0 mV (pH 11.0). As expected, the f-
potential shifted when the pH changed, being positive in the acidic
condition and negative in the neutral and basic conditions.
XRD patterns (Fig. 3) of the Sr-BGNPs after drying at 130 °C (but
prior to thermal stabilization) showed the presence of the cation
precursors. The XRD patterns showed amorphous halos once the
Sr-NPs were heated to 680 °C, indicating that calcium oxide
(CaO) and strontium oxide (SrO), were incorporated into the silica
networks after thermal stabilisation, agreeing with previous stud-
ies on cation incorporation [36,47–50]. Previous slice and view
studies, using FIB and TEM, on 500 nm SiO
2
-CaO NPs made using
the same method, showed a homogeneous distribution of Ca
throughout the NP [36]. As diffusion distances are smaller here,
it is likely that the Sr-BGNPs also have a homogeneous distribution,
although their small size made it impossible to do slice and view
imaging. Sr-BGNPs are likely to have the potential to generate a
sustained release of therapeutic cations because of the Sr being
part of an amorphous silica network.
3.2. Ion release profiles
Fig. 4 shows the dissolution profiles of each of the Sr-BGNPs in
PBS, ALF, and
a
-MEM media. The results demonstrate that the Si
content of the
a
-MEM media, ALF, and PBS increased as the incu-
bation period increased, confirming that soluble silica was released
from the BGNPs at a sustained rate over the immersion periods. Si
release was slowest in PBS, not reaching 20 mg/mL at 3 days. Si
release in ALF and
a
-MEM was faster and exceeded 50 mg/mL at
3 days. In ALF, the concentration of Sr increased sharply during
the first 24 h of immersion, after which it increased more slowly,
Fig. 1. TEM images of Sr-BGNPs: (a) SiO
2
-NPs (100 mol% SiO
2
); (b) 0%Sr-BGNPs (93 mol% SiO
2
, 7 mol% CaO); (c) 6%Sr-BGNPs (87 mol% SiO
2
, 7 mol% CaO, 6 mol% SrO); and (d)
14%Sr-BGNPs (83 mol% SiO
2
, 3 mol% CaO, 14 mol% SrO).
Fig. 2. Dynamic Light Scattering (DLS) results of 0%Sr-BGNPs, 6%Sr-BGNPs and 14%
Sr-BGNPs.
P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80 71
reaching a plateau at 48 h; this increase was significantly higher
for the 14% Sr-BGNPs. In
a
-MEM, the Sr-BGNPs rapidly released
Sr ions during the first 4 h, followed by a gradual release for a pro-
longed period. In PBS, the concentration of Sr increased slightly as a
function of time for the first 24 h of immersion before levelling off.
The medium type and pH affected Sr release: the total amount of Sr
release was greatest in the neutral
a
-MEM medium and was sim-
ilar for the two NP compositions (Table 3), which could be due to
the amino acids present in the medium chelating the Sr
2+
ions. In
contrast, the amount of Sr released in ALF was significantly higher
for the 14% Sr-BGNPs and similar to that measured in
a
-MEM. The
Ca ion concentrations in all three solutions remained approxi-
mately constant over time, after rising in the first 4 h of immersion.
Interestingly, in ALF solution, there was an increase in the Ca ion
concentration for the 6%Sr-BGNPs which was pronounced at 24
and 48 h. It is well known that low pH accelerates cation exchange
in aqueous media for all glasses. The reduction in the stability of
the NPs in an acidic environment is likely to be beneficial by trig-
gering the release of cations inside the lysosomes following cellular
uptake of the NPs. Table 3 showed that the total amount of Ca and
Table 2
Size and Zeta potential of Sr-BGNPs in water at three different pH values: 3.0, 7.4 and 11.0.
Samples DLS size distribution (nm) TEM size (nm) Zeta potential (mV)
pH 3.0 pH 7.4 pH 11.0
0%Sr-BGNPs 89.3 ± 2.9 75.1 ± 5.5 1.5 ± 0.1 22.6 ± 0.6 36.2 ± 0.9
6%Sr-BGNPs 91.2 ± 10.6 79.4 ± 7.7 3.2 ± 2.1 29.7 ± 1.3 32.6 ± 1.6
14%Sr-BGNPs 98.0 ± 9.2 83.2 ± 7.7 4.6 ± 0.3 29.2 ± 0.6 41.6 ± 1.0
Fig. 3. XRD patterns of (a) SiO
2
-NPs; (b) 0%Sr-BGNPs; (c) 6%Sr-BGNPs; and (d) 14%Sr-BGNPs before and after heat treatment process (heating at 680 °C).
72 P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80
Sr release from Sr-BGNPs in ALF and
a
-MEM were 3-times higher
than the amount released in the PBS. None of the Ca profiles show
a reduction in Ca content of the media within the 24 h that the
cells were exposed to the nanoparticles, indicating that there was
no calcium phosphate deposition.
TEM images (Fig. 5) show the morphological changes to the 14%
Sr-BGNPs following incubation in the three different media. When
the 14%Sr-BGNPs were immersed in PBS for 10 days, salt precipita-
tion on the surface of the particles was considerable in PBS (Fig. 5
(a)). Fig. 5(b) shows that in ALF the surfaces of the particles chan-
ged, developing a mottled morphology, indicating that the silicate
network of Sr-BGNPs became more unstable and degraded in the
acidic environment. In
a
-MEM (Fig. 5(c)), a shell with reduced con-
trast was observed around the particles, the particles had a mottled
appearance and necks were observed between the particles, fol-
lowing 24 h of incubation. These features all indicated that the par-
ticles had undergone degradation.
3.3. In vitro cytotoxicity
Our previous work showed that submicron BGNPs can be inter-
nalised by cells and degrade inside the cells [35,43]. Therefore, the
effect of NPs themselves (direct method) was evaluated, in which
the cell monolayer was exposed to NPs directly. A reduction in cell
viability by more than 30% is considered a cytotoxic effect (cell via-
bilities less than 70%) and this was used as a cut-off value to eval-
uate cytotoxicity of these particles (ISO 10993-5). To evaluate the
effect of the 6%Sr-BGNPs and 14%Sr-BGNPs on the viability of the
MC3T3-E1 cells, both NP compositions were introduced to
MC3T3-E1 cells for 24 h (pulse period), i.e. the media containing
non-internalised particles was removed and the cell culture was
continued for 0, 1, 3, and 7 days (chase period). Cells cultured on
tissue culture plates (TCP) served as controls. The results were con-
sistent with our previous report, which showed no toxicity at all
concentrations up to 250 mg/mL, but also little difference between
samples (Fig. 6 (a)). A significant reduction in cell viability was
observed with NP concentrations greater than 500 mg/mL for both
compositions of Sr-BGNPs. When cells were exposed to the Sr-
BGNPs with concentrations ranging from 0.01–250
l
g/mL, the cell
viability did not decrease significantly compared to the control
(TPC). The Stöber SiO
2
-NPs without Ca or Sr did reduce the cell via-
bility after 24 h at a concentration of 200
l
g/mL.
The effect of the BGNP ionic release products were also evalu-
ated (Fig. 6 (b)). The cell viability of the MC3T3-E1 cells increased
Fig. 4. Dissolution profiles of 6%Sr-BGNPs and 14%Sr-BGNPs in three different solutions: (a)
a
-MEM media at pH 7.4; (b) ALF at pH 4.5; (c) PBS at pH 7.4.
Table 3
Percentage elemental release from 6%Sr-BGNPs and 14%Sr-BGNPs after immersion in different media for 1 (
a
-MEM) and 10 days (PBS and ALF).
Samples PBS ALF
a
-MEM
% release
Si Ca Sr Si Ca Sr Si Ca Sr
6%Sr-BGNPs 2.32 1.50 8.17 4.62 7.44 16.13 5.46 3.47 22.94
14%Sr-BGNPs 3.20 0.97 3.68 4.71 3.52 20.38 5.95 4.85 23.56
P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80 73
Fig. 5. Bright-field TEM images of 14%Sr-BGNPs following immersion of the NPs in: (a) PBS for 10 days, (b) ALF for 10 days, and (c)
a
-MEM for 24 h (all scale bars 50 nm).
Fig. 6. Effect of (a) Sr-BGNPs and (b) their ionic release products, on the metabolic cell activity of the pre-osteoblastic cell line (MC3T3-E1) based on an MTT assay after 24 h
pulse followed by chase period in culture of 0, 1, 3, and 7 days (n = 6 per group). The metabolism of the cells treated with different concentrations (0.01–1000
l
g/mL).
*
(6%Sr-
BGNPs) and +(14%Sr-BGNPs) were statistically different from the control (TCP), p < 0.05.
**
(6%Sr-BGNPs) and ++(14%Sr-BGNPs) were statistically decreased from the control
(TCP), p<0.05.
74 P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80
significantly (p<.05) when treated with the Sr-BGNP ion release
products with 87 mol% SiO
2
, 7 mol% CaO, 6 mol% SrO
2
(6% Sr-
BGNPs) and 83 mol% SiO
2
, 3 mol% CaO, 14 mol% SrO
2
(14% Sr-
BGNPs) at 200 and 250 mg/mL after 3 and 7 days, compared to
the cells in the other groups and compared to cells under the basal
condition. The concentration of Si, Ca and Sr ions released into the
media is shown in Supplementary information (Table S2). The con-
centration of Sr required to enhance the activity of the MC3T3-E1
cells was in the range of 2 to 6 mg/mL.
3.4. Osteogenic differentiation
To evaluate therapeutic efficacy of Sr-BGNPs and the ion release
products for bone regeneration, several early and late bone forma-
tion markers were investigated. Fig.7 shows the expression of ALP
activity in the extracellular matrix of the MC3T3-E1 cells treated
with 6%Sr-BGNPs and 14%Sr-BGNPs, and their ionic release prod-
ucts, following 21 days of culture (media contained particles or dis-
solution ions at every media change), compared to cells cultured
under the basal and osteogenic media (basal media plus osteogenic
supplements). There was a significant increase in the ALP activity
of the MCT3T-E1 cells treated with 14%Sr-BGNPs compared to
the 6%Sr-BGNPs in the basal medium. No difference was seen for
the different Sr contents when osteogenic supplements were used.
The ionic release products from both the 6%Sr-BGNPs and 14%Sr-
BGNPs also stimulated the ALP activity of the cultured MC3T3-E1
cells in the basal condition, suggesting that the ALP simulation
resulted from an effect of the ions released from the BGNPs as they
dissolved.
To illustrate the effect of the NPs on osteogenic differentiation
of the MC3T3-E1 cells in vitro, markers associated with early and
late osteogenic differentiation, including Col1a1, OSC and OSP
were investigated using immunohistochemistry (IHC) staining.
Fig. 8 shows that Col1a1, OSC, and OSP were expressed on the
MC3T3-E1 cells following 21 days of culture. In the basal condi-
tion, the late osteogenic differentiation markers, OSC and OSP,
were more overtly expressed by the 14%Sr-BGNPs and their ionic
release products. In contrast, Col1a1 expression was only slightly
enhanced in the basal condition, but was enhanced by the osteo-
genic supplements. Both OSP and Colla1 are proteins associated
with extracellular matrix formation [51,52]. Thus, the data show
that the Sr-BGNPs and their dissolution products could affect both
mineralisation and extracellular matrix formation, but highlight
that osteogenic supplements are also essential for early extracel-
lular matrix formation.
Calcified nodule formation was observed using Alizarin Red S
staining at 7, 14 and 21 days. MC3T3-E1 cells were periodically
incubated with 6% Sr-BGNPs and 14%Sr-BGNPs and their ionic
release products. After 7 days, no difference was observed between
the basal and osteogenic conditions, with either the 6%Sr-BGNPs or
14%Sr-BGNPs or the ionic release products (Fig. 9). After two and
three weeks in culture, mineralization of the MC3T3-E1 cells trea-
ted with 6%Sr-BGNPs and 14%Sr-BGNPs and also their ionic release
products, increased significantly compared with the cells cultured
in the control conditions (basal without BGNPs (Fig. 9(A)) and
osteogenic condition without BGNPs (Fig. 9(B)). This effect of the
BGNPs on mineralisation was significantly more pronounced in
the cells cultured with osteogenic medium. Quantitative analysis
of the Alizarin Red S staining confirmed an increase in calcium for-
mation of the treated MC3T3-E1 cells exposed to both the 6%Sr-
BGNPs and 14%Sr-BGNPs compared to the control cells (untreated
cells in both the basal and osteogenic conditions) (Fig. S3, Supple-
mentary information). This increase in calcium formation was also
seen for the Sr-BGNP ionic release products. These results indicate
that both the 6%Sr-BGNPs and 14%Sr-BGNPs enhanced mineralisa-
tion without the osteogenic supplements, and that this effect can
be attributed to their ionic release products.
The final aim was to track whether BGNPs had been internalised
by the cells and to understand the mechanism by which the BGNPs
were internalised. Cell viability was not affected, remaining above
70% of the positive control, by functionalization of the Sr-BGNPs
with APTES and fluorescently labelling with FITC (Fig. S4, Supple-
mentary information).
Confocal microscopy images (2D in Fig. 10 and z-stacks shown
in the video in Supplementary information) showed that the FITC-
14%Sr-BGNPs were internalised by the cells and were present in
the cytoplasm of the MCT3T-E1 after 24 h of incubation. Fig. 11
shows the fluorescence intensity of the FITC-14%Sr-BGNPs in the
MC3T3-E1 cells, confirming that the NPs were internalised by the
cells. There was a significant decrease in uptake of the FITC-14%
Sr-BGNPs by the cells treated with CytD, suggesting that a
clathrin-dependent endocytosis pathway was responsible for
uptake of the FITC-14%Sr-BGNPs by the MC3T3-E1 cells. However,
some particles were still internalised in the cells treated by CytD
and uptake of FITC-14%Sr-BGNPs was nearly completely sup-
pressed when the MC3T3-E1 cells were treated with all five endo-
cytosis inhibitors, indicating that the MCT3T3-E1 cells also used
alternative pathways to uptake particles. These data indicate that
the FITC-14%Sr-BGNPs enter the cells via a number of endocytosis
pathways.
Fig. 7. Staining for ALP activity, which indicates the differentiation of MC3T3-E1 cells grown in media containing 6%Sr-BGNPs and 14%Sr-BGNPs or their ionic release
products (Sr-BGNP concentration at 250 mg/mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80 75
Fig. 8. Fluorescence image, DAPI (blue), Col1a1, OSC and OSP staining (all green) of MC3T3-E1 cells exposure to 14%Sr-BGNPs and their ionic release products (Sr-BGNP
concentration at 250 mg/mL) in basal and osteogenic conditions (3-week culture period). Scale bar 150 mm. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
76 P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80
4. Discussion
Monodispersed Sr-BGNPs were successfully synthesised using
the modified Stӧber process [36,37]. These nanoparticles were
amorphous and spherical with diameters of 90 ± 10 nm. To
improve the osteogenic response to the BGNPs, additional cations,
such as Sr
2+
were introduced during the sol-gel process [16,53,54].
An important step is to incorporate cations, by calcination above
450 °C, while maintaining particle morphology and diameter
[36,49]. Here, Ca(NO
3
)
2
and Sr(NO
3
)
2
were used as the cation pre-
cursors to incorporate Ca
2+
and Sr
2+
into the silica networks. Dur-
ing drying, Ca(NO
3
)
2
and Sr(NO
3
)
2
deposits onto the secondary
particle surface of the silica [49]. During thermal stabilisation, Ca
and Sr were incorporated into the silica network as network mod-
ifiers, as confirmed by XRD (Fig. 3), without affecting particle size,
distribution and morphology (Table 1, Figs. 1and 2). When the per-
centage of Sr substitution increased from 0 to 14 mol%, the total
amount of network modifiers increased from approximately 7%
to 17%, even though the nominal concentration of potential net-
work modifiers was constant. The reason behind this is that the
ionic radii of Sr (1.12 A°) is larger than Ca (0.99 A°), leading
to a more open silica network, which also leads to more rapid dis-
solution (Fig. 4).
Delivery of Sr inside cells may form part of a therapeutic treat-
ment for osteoporosis, so it is important to understand how the
particles are internalised and the behaviour of the particles in
physiological solutions outside and inside cells. One hypothesis
was that when cells uptake Sr-BGNPs, they form lysosomal vesicles
that encapsulate the particles. Sr-BGNPs would therefore be sub-
jected to acidic conditions inside the cell lysosome. Here, in vitro
dissolution showed that more cations dissolved from the Sr-
BGNPs in ALF solution at pH 4.5 than in the media at pH 7.4,
because cation exchange is promoted under the low pH environ-
ment (Fig. 4). Dissolution in
a
-MEM was more rapid than in PBS
at the same pH, which is likely due to organic molecules having
an affinity (chelation) to cations in the glass.
The in vitro cell viability of MC3T3-E1 cells treated with Sr-
BGNPs was not significantly changed when the particle concentra-
tion increased up to 250
l
g/mL, but Sr-BGNPs concentrations at or
above 500
l
g/mL caused cells death after 3 days in the direct
method. The results suggested BGNPs containing cations had
higher cellular biocompatibility compared to the control of the
much less degradable SiO
2
-NPs (Fig. 6). The relative cell viability
increased when the Sr concentration in the media was in a range
of 2 to 6 mg/mL. Following on from previous research on 45S5 Bio-
glass ionic release products, which induce osteogenic differentia-
tion of osteoblasts without adding osteogenic supplements [8,55–
57], we hypothesised that the media containing the ionic release
products of the Sr-BGNPs would stimulate osteogenic differentia-
Fig. 9. Alizarin red staining for calcified nodule formation from MC3T3-E1 cells
treated with 6% Sr-BGNPs and 14% Sr-BGNPs or their ionic release products (Sr-
BGNP concentration at 250 mg/mL) under: (A) the basal condition and (B) the
osteogenic condition. Scale bar = 400
l
m. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Video
P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80 77
tion of MC3T3-E1 with no added osteogenic supplements. MC3T3-
E1 cells treated under basal conditions with particles or their ionic
release products did indeed show an increase in ALP activity
(Fig. 7) and calcium deposition compared to the control (Fig. 9).
Osteogenesis associated markers, including Col1a1, OSC, and OSP,
were also evaluated (Fig. 8). MC3T3-E1 cells treated with the Sr-
BGNPs and their ionic release products under basal conditions
showed a strong green fluorescent intensity of Col1a1, OSC, and
OSP. The results indicate that Sr-BGNPs stimulate osteogenic dif-
ferentiation and that this effect arises due to the ionic release prod-
ucts of the Sr-BGNPs.
To identify the uptake route used by the Sr-BGNPs, uptake by
MC3T3-E1 cells was investigated under specific inhibitors using
microplate reader and confocal fluorescent microscope, as sug-
gested in previous studies [58,59]. The 14%Sr-BGNPs were proba-
bly predominantly taken up via the clathrin-dependent
endocytosis pathway in the MC3T3-E1 cells (Fig. 11), which is con-
sistent with previous findings that clathrin-dependent endocytosis
is the main pathway for particle size up to 200 nm [59]. However,
the 14%Sr-BGNPs were also internalised into cells treated with all
of the inhibitors. The possible explanation for this might be
because either the concentrations of inhibitors were not suited
Fig. 10. Confocal microscopy images showing internationalisation of the FITC-14%Sr-BGNPs (concentration at 250 mg/mL) by the MC3T3-E1 after pre-treatment with
endocytosis inhibitors; Wor (wortmannin), Ami (amiloride hydrochloride hydrate), Chlor (chlorpromazine), Gen (genistein), CytD (cytochalasin D). Scale bar = 20 mm. Nuclei
were stained with DAPI. F-actin filaments were stained with rhodamine phalloidin. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
Fig. 11. Effect of endocytosis inhibitors on FITC-14%Sr-BGNPs (concentration at 250 mg/mL) internalisation by MCT3T3-E1 cells; Wor (wortmannin), Ami (amiloride
hydrochloride hydrate), Chlor (chlorpromazine), Gen (genistein), CytD (cytochalasin D).
78 P. Naruphontjirakul et al. / Acta Biomaterialia 66 (2018) 67–80
for MC3T3-E1 cells, leading to not completely blocked pathways, or
that the uptake of these particles by the cells occurred through
mixed pathways. The results agree with previous studies on
SiO
2
-CaO NPs in stem cells [35], and it can be speculated that
14%Sr-BGNPs entered into the cells through endocytosis pathways.
The green fluorescent intensity of the confocal microscopy images
(Fig. 10 and the 3D video in Supplementary information) also
showed internalisation of 14%Sr-BGNPs by the MC3T3-E1 cells,
confirming intracellular ion delivery inside the cells.
5. Conclusions
This study reports the osteogenic response of MC3T3-E1 cells
treated with monodispersed Sr-BGNPs SiO
2
-NPs synthesised using
the modified Stöber process and incorporating Ca and Sr. Incorpo-
ration of Ca and Sr into the silica network did not affect the size
and shape of the particles. Culture of the Sr-BGNPs with MC3T3-
E1 cells did not alter the viability of the cells up to concentration
250
l
g/mL. The dissolution products of the Sr-BGNPs were non-
toxic at all concentrations. Ion release products from the 6%Sr-
BGNPs (87 mol% SiO
2
, 7 mol% CaO, 6 mol% SrO) and 14%Sr-BGNPs
(83 mol% SiO
2
, 3 mol% CaO, 14 mol% SrO) at BGNP concentrations
of 200 and 250
l
g/mL enhanced MC3T3-E1 proliferation up to 7
days in vitro. Sr-BGNPs concentrations at or above 500
l
g/mL had
adverse effects on MC3T3-E1 cell viability after 3 days in the direct
method. The 6%Sr-BGNPs and 14%Sr-BGNPs and their ionic release
products had the ability to stimulate an osteogenic response with-
out adding osteogenic supplements in the culture system and this
effect could be attributed to their ionic release products. Crucially,
the data show that the Sr-BGNPs could affect both mineralisation
and extracellular matrix formation, but highlight that osteogenic
supplements are also essential for early extracellular matrix forma-
tion. The results show both an increase in mineralisation and
expression of proteins associated with collagen production. As Sr
content in the NPs and in the dissolution products increased,
ALP, OSC and OSP expression increased. The exact mechanisms
for the cells to uptake Sr-BGNPs remained indefinable, but most
likely based on the clathrin-dependent endocytosis pathway.
These in vitro results revealed that 6%Sr-BGNPs and 14%Sr-BGNPs
at a concentration of 250
l
g/mL promoted osteogenic response
whist maintaining cell proliferation, which is likely to be beneficial
to use as an inorganic drug delivery for bone regeneration
applications.
Acknowledgements
The research work is supported by the Royal Thai Government
and EPSRC (EP/M004414/1) and an Elsie Widdowson Fellowship to
AEP. Raw data is available from rdm-enquiries@imperial.ac.uk.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.actbio.2017.11.008.
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