J Sol-Gel Sci Technol (2011) 58:656–663
DOI 10.1007/s10971-011-2441-8
ORIGINAL PAPER
Effects of acidic catalysts on the microstructure and biological
property of sol–gel bioactive glass microspheres
Bo Lei • Xiaofeng Chen • Young-Hag Koh
Received: 4 February 2011 / Accepted: 2 March 2011 / Published online: 8 March 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Sol–gel bioactive glasses have been developed
for bone tissue regeneration and drug delivery systems as
they have the unique mesoporous structure and high bio-
activity in vitro. To develop more reliable drug delivery
and bone tissue repair systems, it is necessary to control the
morphology and microstructure of bioactive glasses. For
this purpose, bioactive glass microspheres (BGMs) were
prepared by a sol–gel co-template technology using acids
as catalysts. We studied the effects of different acids (citric
acid, lactic acid and acetic acid) on the microstructure and
apatite-forming bioactivity of BGM. The apatite-forming
bioactivity was carried out in simulated body fluid (SBF).
The microstructure and apatite-forming bioactivity of
BGMs were characterized by various methods. Results
showed that acetic acid had little effect on the structure and
bioactivity of BGMs. Differently, the morphology and
microstructure of BGMs could be controlled by changing
citric acid and lactic acid concentrations. In vitro bioac-
tivity test indicated that citric acid and lactic acid derived
BGMs possessed the better apatite-forming capacity than
that derived by acetic acid.
Keywords Bioactive glasses  Microspheres 
Microstructure  Biomaterials  Sol–gel chemistry  Apatite
formation
B. Lei (&)  X. Chen
Biomedical Engineering Institute, South China University of
Technology, Guangzhou 510640, People’s Republic of China
e-mail: leiboaray@yahoo.com.cn
B. Lei  Y.-H. Koh
Department of Dental Laboratory Science and Engineering,
Korea University, Seoul 136703, South Korea
123
1 Introduction
In the fields of tissue regeneration, designing biomaterials
with multifunctional properties such as injectability, drugs
delivery and bioactivity, is one of the most challenging
goals [1–3]. Due to the minimally invasive nature, inject-
able delivery systems have been widely studied and hold
great promise in the fields of drug delivery and tissue
engineering, Moreover, injectable biomaterials can fill
tissue defects of irregular shape in bone regeneration. To
obtain injectable cements, the good flow properties of
particles are necessary to be extruded easily by a syringe.
The enhanced flow properties of micrometer spheres make
it easier to extrude cements than irregularly shaped
cements. As compared to irregularly shaped particles, the
spherical morphology facilitates procedures of bone tissue
regeneration, optimizes drug release and minimizes
inflammatory reactions [4, 5]. Recently, mesoporous silica
and hollow bioceramic microspheres have shown their
potential applications as drug release carriers because of
their large specific surface area, encapsulation properties,
physicochemical and biological advantages [6–8]. How-
ever, for bone tissue regeneration, the application of pres-
ent biomaterials (such as mesoporous silica, polymers and
hydroxyapatite) has been limited because of the lack of
high bioactivity and degradation.
Bioactive glasses (BGs) have been widely used for bone
and dental reconstruction tissue due to their ability to bond
to both soft and hard tissue with minimal inflammation and
toxicity for the host [9, 10]. The sol–gel derived BGs can
be ascribed to be one type of third generation biomaterials,
which are bioactive, restorable and osteoproductive [11].
Menaa et al. have shown that nanoporous sol–gel silica-
based glasses are the idea support for protein bio-encap-
sulation [12]. In addition, the in vitro bioactivity of BGs
J Sol-Gel Sci Technol (2011) 58:656–663
has been demonstrated by the formation rate of crystalline
hydroxycarbonate apatite (HCA) which is the inorganic
part of the human bone, when incubated in a simulated
body fluid (SBF) [13]. Histology taken from BG bone
grafts in human subjects has demonstrated that the surface
activity of the BGs is responsible for their high rates of
bone regeneration [14]. The surface microstructure and
morphology of BGs have an important influence on their
surface activity and cellular response (adhesion, prolifera-
tion and differentiation). Recently, Ostomel et al. and
Vallet-Regı́ et al. developed spherically mesoporous bio-
active glasses by an aerosol-assisted synthesis method [15,
16]. However, the bioactive glass microspheres synthesized
by spray drying possessed a wide particles size distribution
and these particles do not have excellent release properties
as the spray drying caused a major reduction of the surface
area and surface microstructure [17]. Furthermore, ordered
mesoporous bioactive glasses are only produced when the
bioactive glass contains a small amount of CaO and a large
amount of SiO2, while a higher CaO content in the BG
leads to high bioactivity in vitro.
Our previous studies have shown that hydroxyl-carboxyl
acid can control the microstructure of sol–gel BG particles
and improve the biocompatibility of materials [18–21].
Moreover, we also found that polyethylene glycol (PEG)
could induce the formation of sol–gel BG microspheres,
porous particles and nanoscale bulks [22–25]. Therefore,
the objectives of this study were to prepare sol–gel bio-
active glass microspheres (BGMs) by PEG template and
investigate the effects of different acids on their morphol-
ogy, microstructure and apatite-formation bioactivity.
2 Materials and methods
2.1 Materials
Ethanol absolute (EA), hydrochloric acid (HA), citric acid
(CA), lactic acid (LA), acetic acid (AA), tetraethyl ortho-
silicate (TEOS), triethylphosphate (TEP) and calcium
nitrate tetrahydrate (CN) were purchased from Guanghua
Chemical Factory Co., Ltd (Shantou, P.R.China). Poly-
ethylene glycol (PEG6000) was supplied by Sigma–
Aldrich Co (Missouri, USA). All chemical reagents above
were analytical grade. Deionized water (DW) was obtained
from a water purification system (Milipore S.A.S., France).
2.2 Preparation of BGMs
BGMs (mole composition 60%SiO2, 36%CaO, 4%P2O5)
were fabricated by an acid-catalyzed sol–gel co-template
method and HA, CA, LA, and AA were added in this
657
process to control the morphology and microstructure of
BGMs.
First, TEOS (13.5 mL) and TEP (1.4 mL) were mixed
with EA to form a 50 mL solution (solution A). CN (8.5 g),
acids (various weight), and DW (10.0 g) were mixed
together with stirring at room temperature for 30 min
(Solution B). Solution A was added to solution B slowly
and further stirred for 2 h. Then, PEG (1.0 g) was added to
the solution with stirring for 1 h. The resulting sols were
sealed in 100 mL Teflon beakers at 60 °C for 1 day for
aging and gelation. Finally, the gels were removed from
beakers, dried at 60 °C for 24 h, and then heated at 600 °C
(1 °C min-1) for 2 h. The obtained BGM powders were
ground without sieving for testing. The mole ratios of acids
to TEOS and sample codes were shown in Table 1. The
sample without using any organic acid was denoted as
BGM0. Citric acid, lactic acid and acetic acid derived
BGMs were denoted as CBGM, LBGM and ABGM,
respectively.
2.3 Microstructure and in vitro bioactivity analysis
The surface morphology and microstructure were deter-
mined using a JSM6330F high-resolution field emission
scanning electron microscope (FE-SEM) (JEOL, Japan).
Before being observed, the particles were coated with gold
for 5 min under a gas pressure of 50 mTorr. The particle
size distribution was analyzed by a LA-920 laser parti-
cle size analyzer (LPA, Horiba, Japan). The results of
particle size distribution and spherical morphology were
shown in Table 1.
The in vitro bioactivity of materials were tested in a
simulated body fluid (SBF) described by Kokubo et al.
[26]. The particles were soaked in SBF at a concentration
of 1 mg mL-1 in clean polyethylene bottles (200 mL
volume), which had previously been sterilized using 70%
ethanol and washed using deionized water [14]. The bottles
were placed in an incubator at controlled temperature of
37 °C. The pH of the solution was kept at 7.25. All the
samples were soaked in SBF for 24 h without refreshing
solution. Once removed from the incubation, the solids
were separated by centrifugation, then washed three times
with pure ethanol and deionized water, and finally dried at
ambient temperature.
The formation of apatite layer on the surface of samples
was monitored by powder X-ray diffraction (XRD), FE-
SEM and Nicolet Nexus Fourier transform infrared spec-
troscopy (FT-IR) analysis. The XRD experiments were
performed with a Panalytical X’pert PRO diffractometer
equipped with Cu Ka radiation (wavelength 1.548 Å).
XRD patterns were collected in the 2h ranges between 10
and 70° with a step size of 0.02° and counting time of 5 and
10 s per step, respectively.
123
658 J Sol-Gel Sci Technol (2011) 58:656–663

Table 1 Sample codes and characterization summary formation of regular microspheres (Fig. 1; Table 1). Dif-
Sample Acid/TEOS Particle size Spherical
ferently, ABGM shows the irregular morphology at any
codes (molar ratio) distribution (lm) morphology acetic acid concentration and the increase of concentration
can decrease particle size efficiently (Fig. 4; Table 1).
BGM0 0 1–5 Irregular
CBGM1 0.0083 3–5 Irregular
CBGM2 0.017 2–4 Regular 3.2 In vitro bioactivity test
CBGM3 0.033 2–4 Irregular
LBGM1 0.025 1–5 Irregular The apatite-forming bioactivity of samples was tested in
LBGM2 0.05 1–3 Regular simulated body fluid (SBF). Fig. 5 shows the surface
LBGM3 0.1 1–4 Regular morphology and microstructure of BGM0 (a, b), CBGM2
ABGM1 0.025 1–6 Irregular (c, d), LBGM2 (e, f) and ABGM2 (g, h) soaked in SBF for
ABGM2 0.05 1–5 Irregular 24 h. As compared to the images before soaking (Fig. 1),
ABGM3 0.1 0.5–1 Irregular after soaking in SBF for 24 h, the surface morphology of
BGMs has changed and a layer of deposits forms on the
their surfaces. However, the deposit morphology on the
3 Results and discussion surface of BGM was different from each other. Irregular
non-dense particles formed on BGM0 (a, b), a continuous
3.1 Morphology, particle size, and surface layer of dense particles emerged on CBGM2 (c, d),
microstructure analysis LBGM2 (e, f) and ABGM2 (g, h) were visible. Addition-
ally, the grain sizes of deposits on all samples are
By different acid-catalyzed sol–gel process, sol–gel bio- 50–150 nm in length except for CBG0.
active glass microspheres (BGMs) were successfully pro- To determine the remarkable difference of in vitro
duced. Fig. 1 shows the surface morphology and apatite-forming bioactivity for BGMs and CBGM, the rates
microstructure for BGMs derived by different acids at the of apatite deposition on BGM0, CBGM2, LBGM2 and
same concentration. Sol–gel BGM without using organic ABGM2 were tested by FT-IR method. Fig. 6 summarized
acid (BGM0) exhibits the irregular shape, wide particle FT-IR spectra of the samples before and after soaking in
size distribution (1–5 lm) and cracked surface (Fig. 1a, b). SBF for 24 h. Before soaking (Fig. 6a), the spectra are
Acetic acid derived BGM (ABGM2) displays the similar similar and show characteristic absorption bands of Si–O–
feature to BGM0 (Fig. 1g, h). Comparing with BGM0 and Si bonds at 1060 (stretch vibration), 798 (bending vibra-
ABGM2, citric acid and lactic acid derived BGMs tion), and 480 cm-1 (bending vibration). After soaking in
(CBGM2 and LBGM2) show the regularly spherical shape SBF for 24 h, all samples show weak P–O vibrational
and micro-rough surface (Fig. 1c, d, e, f). The particle size bands near 562, 603, 962 cm-1 and the absorption bands of
analysis indicates that using citric acid and lactic acid as carbonate groups at 873 cm-1, indicating the initial pres-
catalysts induced the narrow distribution range (Table 1). ence of poor crystalline apatite layer [27].
Except for acid species, the acid concentration is the The formation of the crystalline apatite layer after
other important factor influencing the morphology and immersing in SBF was also demonstrated by XRD analysis
structure of BGMs. Figs. 2, 3 and 4 reveal the effects of (Fig. 7). Before soaking (a), only one small wide peak
citric acid, lactic acid, and acetic acid concentrations on the between 20 and 30°was detected for all samples. This wide
morphology and structure of BGMs, respectively. The peak indicates representative amorphous nature of bioac-
results show that the morphology and size of microspheres tive glass [15]. After being soaked in SBF for 24 h, all
were correlative with the acid concentrations. As shown in samples show the formation of an apatite phase on their
Fig. 2 and Table 1, at lower and higher concentration for surfaces. New visible peaks at 26, 32, 39, 46, 49, 53° can
citric acid, BGM presents the irregular morphology be ascribed to (002), (211), (310), (222), (213), and (004)
(CBGM1 and CBGM3). BGM shows the regularly spher- reflections of hydroxyapatite (HA) (PDF number 09-0432).
ical shape at certain citric acid concentration (CBGM2). The presence of these characteristic peaks suggests the
Increasing citric acid concentration produced the narrow formation of crystalline HA for all samples.
particle size distribution of BGMs. Compared to CBGM; Based on the analysis of FESEM, LPA, FT-IR and
LBGM exhibits a wide particle size distribution because of XRD, it is should be noted that citric and lactic acids-based
aggregation at lower or higher lactic acid concentration catalysis, together with PEG template, can induce the for-
(Fig. 3). Proper lactic acid concentration can induce the mation of relatively dispersive and regularly shaped BGM

123
J Sol-Gel Sci Technol (2011) 58:656–663 659

Fig. 1 FE-SEM images of acid catalyzed BGMs, a, b BGM0, c, d CBGM, e, f LBGM, g, h ABGM at different magnification

123
660
Fig. 2 Micrographs of CBGM1 (a, b) and CBGM3 (c, d) at different magnification
which also showed the high deposit capacity of HA. The
formation mechanism of microspheres with regular mor-
phologies can be explained by combination functions of
organic acids and polymer template.
The sol–gel process producing BG involves the hydro-
lysis of TEOS using acid as a catalyst and different types of
acids have different influences on the structure of BG.
Firstly, the precursors of bioactive glass such as TEOS,
TEP, and CN hydrolyzed under the catalysis of acids and
formed the clear sols. In this sol, citric acid and lactic acid
were absorbed on the surface of BG sol particles by the
hydroxyls and carboxyl (in molecular structure) interac-
tion. In this process, organic acids are not only catalysts but
also stable agents of BG sol particles. PEG is the water-
soluble polymer and contains many hydroxyls in its
structure. When PEG was added in the BG sols, it will
prevent the aggregation of sol particles as a dispersant [22].
Mono-dispersible microspheres formed through the phase
separation process after aging and gelation. If no PEG exits
in the sol, the phase separation will not appear and no
dispersible microspheres form.
Our previous studies have shown that hydroxyl-carboxyl
acids can control the surface structure of sol–gel BG
123
J Sol-Gel Sci Technol (2011) 58:656–663
particles depending on the hydrogen bond interaction [18,
21]. BG presents the incomplete hydrolysis under natural
condition (without any acid), thus BGM0 exhibits the
irregularly shaped structure with a cracked surface
(Fig. 1a, b). Citric and lactic acids, as hydroxyl-carboxylic
acids and weak organic acids, may present the silicate
particles growth and induce the homogeneous hydrolysis of
precursor by hydrogen bonding interactions with the inor-
ganic precursor [28]. As a result, regularly shaped micro-
spheres with the relatively smooth surface formed under
the catalysis of citric and lactic acids (Fig. 1c, d, e, f).
Comparing with citric and lactic acids, acetic acid is a
relatively weak organic acid without hydroxyl in its
structure, which induces the inhomogeneous hydrolysis of
BG and produced the irregularly shaped structure.
The results of apatite-forming bioactivity test showed
that all samples induce the formation of crystalline apatite
layers in 24 h, showing the high apatite-forming bioactiv-
ity. It was indicated that addition of different organic acids
changed the morphology and microstructure of micro-
spheres, but did not weaken their apatite-forming capacity.
As compared to irregular shaped particles, the spherical
morphology facilitates procedures of bone tissue
J Sol-Gel Sci Technol (2011) 58:656–663 661

Fig. 3 Micrographs of LBGM1 (a, b) and LBGM3 (c, d) at different magnification

Fig. 4 Micrographs of ABGM1 (a, b) at different magnification and ABGM3 (c)

regeneration, optimizes drug release and minimizes
inflammatory reactions [4, 5]. In this study, the use of citric
acid and lactic acid in the sol–gel process makes it possible
to control spherical morphology and structure of BGs as
well as their in vitro apatite-forming bioactivity.
4 Conclusions
In our investigation, the effects of organic acids (including
kinds and concentration) on the morphology, surface
microstructure and apatite-forming bioactivity of BGMs
have been studied. The results showed that the addition of
citric acid and lactic acid could produce formation of
regularly shaped microspheres with relatively smooth sur-
face, while not changing the size of microspheres. Regular
bioactive glass microspheres did not form if there was no
acid or acetic acid in the sol–gel process. The particle size
decreased with the increase of acetic acid concentration. In
addition, the apatite-forming bioactivity test exhibited that
all samples induced the formation of apatite in SBF for
24 h.

123
662 J Sol-Gel Sci Technol (2011) 58:656–663

Fig. 5 Surface morphology of BGM0 (a, b), CBGM2 (c, d), LBGM2 (e, f), ABGM2 (g, h) after soaking in SBF for 24 h

123
J Sol-Gel Sci Technol (2011) 58:656–663
Fig. 6 XRD patterns of (a) sample before soaking in SBF, (b) BGM0
soaked in SBF for 24 h, (c) CBGM2 soaked in SBF for 24 h,
(d) LBGM2 soaked in SBF for 24 h, (e) ABGM2 soaked in SBF for 24 h
Fig. 7 XRD patterns of (a) sample before soaking in SBF, (b) BGM0
soaked in SBF for 24 h, (c) CBGM2 soaked in SBF for 24 h,
(d) LBGM2 soaked in SBF for 24 h, (e) ABGM2 soaked in SBF for 24 h
Acknowledgments The research was supported by National Natural
Science Foundation of China (Grant No. 51072055), the key project
of the National Natural Science Foundation of China (Grant No.
50830101) and the National 973 project of China (2011CB606204).
663
References
1. Causaa F, Nettib PA, Ambrosio L (2007) Biomaterials 28:5093
2. Kretlow JD, Klouda L, Mikos AG (2007) Adv Drug Deliv Rev
59:263
3. Vallet-Regı́ M, Balas F, Colilla M, Manzano M (2008) Prog Solid
State Chem 36:163
4. Manzano M, Aina V, Are0 an CO, Balas F, Cauda V, Colilla M
(2008) Chem Eng J 137:30
5. Paul W, Sharma CP (1999) J Mater Sci Mater Med 10:383
6. Slowing II, Trewyn BG, Giri S, Lin VSY (2007) Adv Funct
Mater 17:1225
7. Komlev VS, Barinov SM, Girardin E, Oscarsson S, Rosengren A,
Rustichelli F (2003) Sci Technol Adv Mater 4:503
8. Huang W, Mohamed NR, Delbert ED, Brad AM (2009) J Mater
Sci Mater Med 20:123
9. Hench LL (1997) Curr Opin Solid State Mater Sci 2:604
10. Clupper DC, Hench LL, Mecholsky JJ (2004) J Euro Cera Soc
24:2929
11. Vallet-Regi M, Ragel CV, Salinas AJ (2003) Euro J Inorg Chem
6:1029
12. Menaa B, Menaa F, Aiolfi-Guimaraes C, Sharts O (2010) Int J
Nanotechnol 7:1
13. Hench LL, Polak JM (2002) Science 295:1014
14. Zhong J, Greenspan DC (2000) J Biomed Mater Res 53:694
15. Ostomel TA, Shi QH, Tsung CK, Liang HJ, Stucky GD (2006)
Small 2:1261
16. Arcos D, López-Noriega A, Ruiz-Hernández E, Terasaki A,
Vallet-Regı́ M (2009) Chem Mater 21:1000
17. Radin S, Chen T, Ducheyne P (2009) Biomaterials 30:850
18. Chen X, Lei B, Wang Y, Zhao N (2009) J Non-Cryst Solids
355:791
19. Lei B, Chen X, Wang Y, Zhao N, Du C, Fang L (2009) J Non-
Cryst Solids 355:2583
20. Lei B, Chen X, Wang Y, Zhao N, Du C, Fang L (2010) J Biomed
Mater Res 94A:1091
21. Lei B, Chen X, Wang Y, Zhao N, Du C, Fang L (2010) Biomed
Mater 5:4103
22. Lei B, Chen X, Wang Y, Zhao N (2009) Mater Lett 63:1719
23. Lei B, Chen X, Wang Y, Zhao N, Miao G, Lin C (2010) Mater
Lett 64:2293
24. Lei B, Chen X, Wang Y, Zhao N, Du C, Fang L (2010) J Am
Ceram Soc 93:32
25. Lei B, Chen X, Wang Y, Zhao N, Du C, Fang L (2009) J Non-
Cryst Solids 355:2678
26. Kokubo T, Takadama H (2006) Biomaterials 27:2907
27. Notingher I, Jones JR, Verrier S, Bisson I, Embanga P, Edwards
P, Polak JM, Hench LL (2003) Spectroscopy 17:275
28. Izutsu H, Mizukami F, Sashida T, Maeda K, Kiyozumi Y, Ak-
iyama Y (1997) J Non–Cryst. Solids 212:40
123