Bioactive glasses

DOI: 10.1002/smll.200600177
Spherical Bioactive Glass with Enhanced
Rates of Hydroxyapatite Deposition and
Hemostatic Activity**
Todd A. Ostomel, Qihui Shi, Chia-Kuang Tsung,
Hongjun Liang, and Galen D. Stucky*
Bioactive glasses, which can be described by the general
chemical formula SiO
2
CaOP
2
O
5
MO (M=Na, Mg, etc.),
are used for bone and dental reconstruction because of their
ability to bond to both soft and hard tissue with minimal in-
flammation and toxicity for the host.
[1, 2]
Although the medi-
cal application of oxide materials has been primarily for
load-bearing implants,
[3]
there is a recent interest in porous
inorganic materials that can interface with soft biological
tissue/fluid,
[4, 5]
for example, blood. There is a widespread
need for the development of new materials that can be ap-
plied by first-aid responders to prevent lethal amounts of
blood loss from traumatically injured individuals.
[6]
Massive
hemorrhage accounts for over 50% of battlefield fatalities,
and consequently the United States military has expended
great efforts to develop rapid-acting hemostatic agents, the
leader of which is a zeolite-based
[7]
composite that can be
poured onto a wound to stabilize a victim who might other-
wise bleed to death.
[8]
Our laboratory was the first to identi-
fy hemostatic bioactive glass as a rapid-acting hemostatic
agent
[9]
that demonstrates clotting activity comparable to
the zeolite-based hemostatic agent currently used by United
States soldiers but without the negative thermal side effect
that tended to burn healthy tissue during application. He-
mostatic bioactive glass, as a function of its Si/Ca ratio, high
surface area, and porosity, is distinct from the traditional bi-
oglass used for bone repair.
[10, 11]
Because both biominerali-
zation
[12]
and blood clotting
[13]
are surface-mediated phe-
nomena, we investigated the effect of changing the shape of
the bioactive glass particles on the in vitro rates of hydrox-
yapatite deposition and hemostatic efficacy. High-surface-
area inorganic oxides are attractive materials for bone and
tooth repair agents and now as newly identified hemostatic
agents. Both the rates of hydroxyapatite deposition and he-
mostatic activity are enhanced for spherical bioactive glass
particles compared to the analogous irregular bioactive
glass. The porosity, Si/Ca ratio, and morphology of mesopo-
rous bioactive glass microspheres (MBGMs) can be tailored
to elicit a predictable biological response and they are
therefore suitable for a variety of wound-healing scenarios.
We have previously reported the preparation of high-
surface area mesoporous bioactive glass particles that dem-
onstrated accelerated deposition rates of hydroxyapatite in
simulated body fluids (SBFs).
[14]
This synthesis relied on
solgel chemistry techniques in which Si, Ca, and P inorgan-
ic precursors (tetraethyl orthosilicate, calcium nitrate, and
triethyl phosphate) were controllably condensed around a
structure-directing template (P123, triblock copolymer;
(polyethylene oxide)
20
(polypropylene oxide)
70
(polyethylene
oxide)
20
). Despite the encouraging in vitro deposition rates
of hydroxyapatite on mesoporous bioactive glass, the evapo-
ration-induced cooperative-assembly process
[10, 15, 16]
is time
consuming and requires a final grinding step to prepare the
fine powder consistency of irregular-shaped bioactive glass
that was used to formulate cements.
Cooperative assembly of amphiphilic molecules (e.g. ,
surfactants and block copolymers) within aerosols is a pow-
erful and flexible method for synthesizing ordered mesopo-
rous silica particles, which can be used for catalysis, con-
trolled drug release, separation, and energy conversion.
[1719]
We have extended our previous solgel preparation of mes-
oporous bioactive glass
[14]
to incorporate this aerosol
method for the preparation of spherical bioactive glass par-
ticles. The P molar content was maintained at 4% for all
samples; the 80 in MBGM-80 represents the Si molar
content; Ca content =100P%Si %. Ethanol sols of the
inorganic precursors and structure-directing surfactant were
atomized in an N
2
carrier gas and passed through a 4008C
horizontal tube furnace. The nonvolatile components of the
sol were enriched at the nitrogen/liquid interface of the aer-
osol droplet, which resulted in the cooperative assembly of
the liquid-crystalline mesophases. The spherical particles
were collected on a membrane filter as they exited the hori-
zontal tube furnace and subsequently calcined at 5508C to
remove the surfactant. This continuous aerosol method re-
duces the synthesis time of MBGMs to a matter of seconds.
More importantly, the rate of hydroxyapatite deposition in
SBF and hemostatic activity is greatly enhanced for the
MBGMs compared to previous bioactive glass formulations.
MBGMs are spherical in morphology, and free of de-
fects and macropores, and have diameters ranging from
100 nm to 1 mm (Figure 1a). The particle size distribution
and mean average size can be tuned by controlling the solu-
tion composition and processing parameters (e.g. , gas flow
rate, furnace temperature, or use of a vibrating orifice aero-
sol generator
[18]
). Calcined MBGMs have a uniform meso-
structure with no apparent long-range order (Figure 1b).
Fourier-transform infrared (FTIR) spectra confirm the pres-
ence of SiO groups, as evidenced by the peaks at 1085 and
802 cm
1
, and amorphous phosphate groups as evidenced by
[*] Dr. T. A. Ostomel, Q. Shi, C.-K. Tsung, Dr. H. Liang,
Prof. G. D. Stucky
Department of Chemistry and Biochemistry
University of California, Santa Barbara, CA 93106 (USA)
Fax: (+1) 805-893-4120
E-mail : stucky@chem.ucsb.edu
[**] This work was funded by the Office of Naval Research, ONR
Awards N00014-04-1-0654 and N00014-06-1-0145, and made use
of the MRL central facilities supported by the MRSEC Program of
the National Science Foundation under Award No. DMR05-20415.
TAO and QS contributed equally to this work. Supporting Infor-
mation Available: Synthesis of mesoporous bioactive glass
microspheres and cements, materials characterization including
SEM, TEM, BET, and XRD. Thromboelastograph clotting parame-
ters.
Supporting information for this article is available on the WWW
under http://www.small-journal.com or from the author.
small 2006, 2, No. 11, 1261 1265 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 1261
the peaks between 600570 cm
1
(see Supporting Informa-
tion, Figure S3).
[2]
BrunauerEmmettTeller (BET) N
2
-ad-
sorption/desorption isotherms of the calcined MBGM are
representative of type IV isotherms, with an H2-type hyste-
resis loop corresponding to bottlelike pores (see Supporting
Information, Figure S4). The calculated BET surface area,
pore volume, and pore size for MBGM-80 and MBGM-60
are 386 m
2
g
1
, 0.70 cm
3
g
1
, and 6.5 nm, and 393 m
2
g
1
,
0.77 cm
3
g
1
, and 6.9 nm, respectively. The spherical mor-
phology, large pore volume, and specific surface area will fa-
cilitate cation release, which is believed to be crucial to
both hydroxyapatite deposition and contact-activated coagu-
lation.
For precise bone- and dental-repair applications, trauma
physicians would prefer to work with bone-generating bio-
active glass as injectable cements rather than as loose pow-
ders. We have prepared MBGM cements for bone and
dental repair by mixing MBGM with an ammonium phos-
phate buffer solution. The cement is soft enough to be ex-
truded by a syringe and sets in %8 min (under standard tem-
perature and pressure conditions) due to the precipitation
of hydroxyapatite nanocrystalline linkers between the
MBGMs. MBGM cements are malleable before setting and
retain their shape and mechanical strength without crum-
bling after setting. This mechanical property is essential for
repairing irregularly shaped bone and dental defects. The
enhanced flow properties of submicrometer spheres make it
easier to extrude MBGM cements rather than irregular bio-
active glass cements.
It is believed that the biocompatibility of bone-generat-
ing bioactive glass implants is due in part to the materials
ability to nucleate new biologically active layers of hydroxy-
apatite at the implanttissue interface.
[20]
The in vitro assess-
ment of in vivo bone-generating bioactivity is typically con-
ducted by monitoring the formation of hydroxyapatite on
the surface of bioactive glass after immersion in SBF. After
mixing the MBGMs with the ammonium phosphate buffer
solution, weak X-ray diffraction (XRD) peaks at 2q=268
(002) and 328 (211) corresponding to hydroxyapatite are ob-
served. The broad peak at 2q=238
is due to the amorphous nature of
the bioactive glass walls (Figure 2).
Bone generation: Despite efforts
to accelerate the kinetics of
hydroxy apatite deposition by tailor-
ing the compositional or textural
properties of bioactive glass,
[10, 15, 21]
the reported induction period of
hydroxyapatite crystallization in
SBF is !24 h. After only 1 h of im-
mersion in SBF both MBGM-60
and MBGM-80 cements demon-
strated increased (002) and (211)
X-ray diffraction peak intensities,
as well as the presence of higher-
order hydroxyapatite reflections.
Comparable hydroxy apatite crystal-
linity was observed on irregularly shaped bioactive glass
after about one day.
[14]
Scanning electron microscopy (SEM)
images of the MBGM cements demonstrate a uniform cov-
erage of hydroxyapatite after 1 h of immersion in SBF (Fig-
ures 3ac). Transmission electron microscopy (TEM)
images of MBGM cements reveal the presence of rodlike
crystals that are confirmed by selected-area electron diffrac-
tion (SAED) to be hydroxyapatite (Figure 3d and Support-
ing Information Figure S5). Energy-dispersive X-ray analy-
sis demonstrates that the hydroxyapatite has an average Ca/
P molar ratio of 1.4. This calcium-deficient formulation of
hydroxyapatite, which can be represented by the general
Figure 1. a) Scanning electron microscopy (SEM) image of MBGM-80 after calcination. b) Transmis-
sion electron microscopy (TEM) image of MBGM-80 after calcination.
Figure 2. Wide-angle XRD patterns of a) MBGM-80 cement, b) MBGM-
60 cement, c) MBG-80 cement soaked in SBF for 1 h, d) MBGM-60
cement soaked in SBF for 1 h.
1262 www.small-journal.com 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim small 2006, 2, No. 11, 1261 1265
communications
formula Ca
10x
(HPO
4
)
x
(PO
4
)
6x
(OH)
2x
(1.33 x 1.67), is
known to catalyze the deposition of new layers of hydroxya-
patite faster than stoichiometric hydroxyapatite (x=0) .
[22]
We believe that the rapid in vitro bone-forming bioactiv-
ity of MBGM cements is due to the calcium-deficient hy-
droxyapatite that precipitates during the setting period. Cal-
cium-deficient hydroxyapatite is known to assist in the im-
mediate nucleation and growth of biologically equivalent
apatite when immersed in simulated body fluid; stoichio-
metric hydroxyapatite requires at least 15 h, and sometimes
up to several months, for the same crystal growth. The
bone-forming activity of stoichiometric hydroxyapatite is
less than that of bioactive glass,
[23]
but can be greatly en-
hanced by mixing the two materials to form a biphasic com-
posite.
[24]
When immersed in simulated body fluid, stoichio-
metric hydroxyapatite, bioactive glass, and the biphasic-bio-
active glass/stoichiometric-hydroxyapatite composite were
observed to catalyze the precipitation of hydroxyapatite in
45 d, 7 d, and 12 h, respectively. The deposition of new hy-
droxyapatite layers on bioactive glass improves biocompati-
bility by promoting cellular processes.
[25]
The calcium-defi-
cient hydroxyapatite/MBGM cement described in this
report demonstrates faster deposition rates of secondary hy-
droxyapatite layers from SBF than the previously men-
tioned examples, a good indication of its potential for
in vivo bone-forming bioactivity.
Previous reports monitoring the in vitro deposition of
hydroxyapatite on bioactive glass composites in SBF
[26]
have
shown that, in general, there is an increase in the pH and
concentration of Si
IV
and Ca
II
species, whereas the P
V
con-
centration in SBF decreas-
es. The dissolution of Ca
II
species from bioactive
glass, and exchange with
H
3
O
+
, favors the forma-
tion of silanol groups while
simultaneously increasing
the local Ca
II
concentra-
tion beyond the supersatu-
ration point and inducing
crystal nucleation.
[27]
Meso-
porous bioactive glass
composites are reported to
increase the local Ca
II
con-
centration faster than non-
porous bioactive glass
composites, due to the in-
creased surface area in
contact with the solution,
although nearly all of the
Ca
II
species are consumed
at the expense of forming
calcium-deficient hydroxy-
apatite at the bioactive
glass interface. In similar
experiments to ours, Zhao
and co-workers
[10]
reported
that the local Ca
II
concen-
tration maximizes at
%200 ppm shortly after immersion of bioactive glass in SBF
and steadily decreases thereafter.
The MBGM cements presented in this report are unique
in that they contain calcium-deficient hydroxyapatite nano-
crystals, as a consequence of mixing the bioactive glass mi-
crospheres with the ammonium phosphate buffer, prior to
immersion in SBF. We found that bioactive glass samples
with a low Si/Ca ratio (i.e. , BG60) are observed to have
faster deposition rates of hydroxyapatite,
[14]
in agreement
with commercially marketed bioactive glass compositions.
[3]
The MBGM cements that possess higher Ca loadings are
observed to initially contain larger crystals of calcium-defi-
cient hydroxyapatite than MBGM cements with a lower Ca
loading (i.e. , MBGM-60 cements versus MBGM-80 ce-
ments). The bioactive glass cements, which are immersed in
SBF, are composed of both the calcium-deficient hydroxy-
apatite crystals as well as unreacted bioactive glass material
that will provide Ca
II
species and reactive silanol groups to
catalyze further hydroxyapatite nucleation and growth.
Rapid-acting hemostatic agent: Mammalian red blood
cells are programmed for death after passing through the
heart on average 210
5
times
[28]
( %100 d), and are constant-
ly being regenerated from stem cells that differentiate
within the bone marrow. Despite the intimate contact be-
tween blood and bone materials, the inorganic component
of which is biological apatite, there is still much to be learnt
about the interfacial phenomena that occur between cellular
surfaces and oxide materials.
Porous hemostatic bioactive glass is an ideal rapid-acting
hemostatic agent because this material releases Ca
II
ions
Figure 3. a) SEM of MBGM-80 cement (bar =2 mm). b,c) SEM of MBGM-80 cement soaked in SBF for 1 h
(bar =10 mm, 2 mm, respectively). d) TEM of MBGM-80 cement soaked in SBF for 1 h.
small 2006, 2, No. 11, 1261 1265 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.small-journal.com 1263
upon hydration, is able to concentrate blood constituents
through capillary absorption of fluid-phase media, and is
composed of an insoluble inorganic core that could provide
an effective support for thrombosis. The in vitro hemostatic
activity of MBGM was evaluated using a thromboelasto-
graph, a clinical instrument that monitors the change in vis-
coelasticity of blood during clot formation.
[5, 29]
A polyeth-
ylene cup, containing blood and a hemostatic agent, is rotat-
ed 58 about a torsion wire. The time until amplitude of
the bimodal symmetric viscoelasticity curve is 2 mm is re-
ferred to as R (minutes), and represents the initial detection
of clot formation. The angle between the tangent to the
curve and the horizontal is referred to as a (8), and is relat-
ed to the rate of coagulation. The maximum separation of
the curves is referred to as MA (dyncm
2
10
5
Ncm
2
) and
represents the maximum clot strength.
MBGMs are hemostatically active and rapidly promote
blood-clot formation. The time until clot detection decreas-
es linearly with the amount of hemostatic agent added to
the blood, and correspondingly the rate of coagulation in-
creases with the amount of hemostatic agent added
(Figure 4). These results demonstrate that the fastest con-
tact-activating agents are those bioactive glass composites
with a high Si/Ca ratio (R
MBGM-80
<R
MBGM-60
). Hemostatic
bioactive glass materials can perform the dual role of sup-
plying calcium ions, which are cofactors through the blood-
clotting cascade, and under physiological conditions provide
a negatively charged siliceous oxide as a support for sur-
face-dependent thrombotic reactions; there will thus be an
optimum ratio of SiO
2
to CaO in bioactive glass for the fast-
est hemostatic response. Porous MBGMs demonstrate re-
duced clot detection times and increased coagulation rates
compared to nonporous MBGMs. Porous materials concen-
trate fluid-phase media in blood by capillary absorption,
and this concentrating effect promotes coagulation.
[30]
Al-
though all blood clots induced by MBGM materials were
stronger than clots formed in their absence (MA
MBGM
!
50 dyncm
2
), there is no relation between the ultimate
strength of the induced blood clot and the amount of mate-
rial added for the mass range studied.
Ca content is a critical material parameter to consider
when designing wound-healing agents because of the ubiqui-
tous role of Ca
II
ions throughout the cascade of reactions
that lead to fibrin polymerization and clot stabilization.
[13, 31]
Our previous studies with irregular bioactive glass
[9]
re-
vealed enhanced procoagulant activity with an increasing Si/
Ca ratio, suggesting that even the more Si-rich bioactive
glass compositions delivered sufficient amounts of Ca
II
spe-
cies to induce an accelerated coagulation response. This
report further confirms the increased procoagulant activity
associated with bioactive glasses as the ratio of Si/Ca is in-
creased as well as describes the inverse relationship between
the Si/Ca ratio in bioactive glass and the corresponding
bone-generating properties. The bioactive glass samples
with the greatest bone-forming activity are those with a low
Si/Ca (BG60) in contrast with the high Si/Ca (BG80) bioac-
tive glass samples, which demonstrated the greatest hemo-
static activity. We have reported that hydroxyapatite, which
is similar to bioactive glass without any SiO
2
and represents
the extreme low end of the Si/Ca range, exhibits antithrom-
botic activity.
[9]
We find that those bioactive glass composi-
tions that are best for fast bone growth (low Si/Ca, e.g. ,
MBGM-60) are the least active towards eliciting a rapid
contact-activated coagulation response. Those bioactive
glass compositions that demonstrate the most rapid contact-
activated clotting response (high Si/Ca, e.g., MBGM-80) are
also the compositions with the least activity for bone gener-
ation. We are studying whether it is the greater bioavailabil-
ity of Ca
II
ions at the wound site that promotes coagulation
and hydroxyapatite nucleation or whether the surface of a
Ca-containing oxide is the active catalytic surface for these
and other related processes. In light of the complexity of
the blood coagulation cascade,
[32]
it is most likely that the
Ca
II
ions supplied by bioactive glasses have multiple
accelera tory functions.
Keywords:
bioactivity biocompatible materials glasses
hydroxyapatite microspheres
Figure 4. a) Thromboelastograph plot of the hemostatic activity
MBGM-80 induced coagulation vs. blood without MBGM-80. b) Plot
of both clot detection time, R, (filled shapes) and rate of coagulation,
a, (unfilled shapes) vs. the amount of mesoporous bioactive micro-
spheres. Data represents the mean of four trials. &=MBGM-60,
*=MBGM-80, ~=MBGM-60 nonporous, !=MBGM-80 nonpo-
rous, + =sheep blood without MBGM.
1264 www.small-journal.com 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim small 2006, 2, No. 11, 1261 1265
communications
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Received: April 14, 2006
Revised: July 10, 2006
small 2006, 2, No. 11, 1261 1265 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.small-journal.com 1265