1. J Mater Sci: Mater Med
The inﬂuence of phosphorus precursors on the synthesis
and bioactivity of SiO2–CaO–P2O5 sol–gel glasses and glass–
Renato Luiz Siqueira • Edgar Dutra Zanotto
Received: 12 June 2012 / Accepted: 15 October 2012
Ó Springer Science+Business Media New York 2012
Abstract Bioactive glasses and glass–ceramics of the
SiO2–CaO–P2O5 system were synthesised by means of a sol–
gel method using different phosphorus precursors according
to their respective rates of hydrolysis—triethylphosphate
(OP(OC2H5)3), phosphoric acid (H3PO4) and a solution
prepared by dissolving phosphorus oxide (P2O5) in ethanol.
The resulting materials were characterised by differential
scanning calorimetry and thermogravimetry, X-ray diffraction, Fourier transform infrared spectroscopy, scanning
electron microscopy coupled with energy dispersive X-ray
spectroscopy and by in vitro bioactivity tests in acellular
simulated body ﬂuid. The different precursors signiﬁcantly
affected the main steps of the synthesis, beginning with the
time required for gel formation. The most striking inﬂuence
of these precursors was observed during the thermal treatments at 700–1,200 °C that were used to convert the gels into
glasses and glass–ceramics. The samples exhibited very
different mineralisation behaviours; especially those prepared using the phosphoric acid, which had a reduced onset
temperature of crystallisation and an increased resistance to
Electronic supplementary material The online version of this
article (doi:10.1007/s10856-012-4797-x) contains supplementary
material, which is available to authorized users.
R. L. Siqueira (&)
Grupo de Pesquisas em Nanotecnologia e Nanomateriais,
Centro Federal de Educacao Tecnologica de Minas Gerais,
Campus Timoteo, Av. Amazonas 1193, Vale Verde,
Timoteo, MG 35183-006, Brazil
R. L. Siqueira Á E. D. Zanotto
Laboratorio de Materiais Vıtreos, Departamento de Engenharia
de Materiais, Universidade Federal de Sao Carlos,
Rod. Washington Luıs km 235, CP 676, Sao Carlos,
SP 13565-905, Brazil
devitriﬁcation. However, all resulting materials were bioactive. The in vitro bioactivity of these materials was
strongly affected by the heat treatment temperature. In
general, their bioactivity decreased with increasing treatment temperature. For crystallised samples obtained above
900 °C, the bioactivity was favoured by the presence of two
crystalline phases: wollastonite (CaSiO3) and tricalcium
Melt-quenched silicate glasses containing calcium, phosphorus and alkali metals, such as the quaternary glass
SiO2–CaO–Na2O–P2O5 system, have been extensively
studied for their remarkable interactions with living tissues,
since they were discovered by Hench . In addition to
their bioactivity, i.e., their ability to form in situ
hydroxyapatite (HA) layers on their surfaces, which promotes a strong interfaces and strong bonds to cartilage and
bones, it has been demonstrated that some bioactive glasses
affect osteoblast activity and upregulate at least seven
families of genes when primary human osteoblasts are
exposed to their ionic dissolution products; such genes
include those that encode proteins associated with osteoblast proliferation and differentiation [1, 2].
The use of sol–gel processing to prepare bioactive
glasses began in the late 1980s with the work of Li et al.
, which led to a patent in 1991 concerning the production of the ﬁrst alkali-free bioactive glass compositions .
These authors demonstrated that the bioactive gel-glasses
of the ternary SiO2–CaO–P2O5 system exhibited in vitro
bioactivity, even for compositions containing approximately 90 mol% SiO2. The properties observed in the
SiO2–CaO–P2O5 sol–gel glasses are entirely opposite those
2. J Mater Sci: Mater Med
observed for glasses prepared by traditional melt-quenching methods, which tend to be chemically stable and
lacking in bioactive properties due to their signiﬁcantly
high SiO2 contents [1, 3, 5, 6]. In addition to their intrinsically high speciﬁc surface areas (approximately
200 m2 g-1), which signiﬁcantly increase the rate of HA
layer formation and, consequently, the bioactivity, the
surfaces of the bioactive gel-glasses can be functionalised
by a variety of surface-chemistry methods due to their
numerous existing silanol groups (Si–OH) [3, 7–12].
It is important to note that this ﬁnding offers a potential
processing method for molecular and textural tailoring of
the biological behaviour of a new class of bioactive
materials. This method has facilitated the development of
structures similar to that of trabecular bone (a spongy,
highly porous form of bone tissue), thereby promoting
sufﬁcient access to a wide range of complex bioactive
material conﬁgurations, such as scaffolds suitable for tissue-engineering applications [13, 14]. Additionally, it is
possible to signiﬁcantly improve the mechanical performance of these materials via their glass–ceramic transformations by means of a controlled crystallisation process
For all these reasons, sol–gel processing has been widely
investigated for the preparation of bioactive glasses and
glass–ceramics. For instance, many new systems and
compositions have emerged following the use of sol–gel
processing, and these systems often contain other elements,
such as Zn, Mg, Ag, Sr, and Sm [7, 21–32]. More recently,
systems containing Na and possessing compositions similar
to BioglassÒ 45S5 and BiosilicateÒ also have been reported
[33–35]. These materials are of the same quaternary SiO2–
CaO–Na2O–P2O5 system and also show very high bioactivity in both in vitro and in vivo conditions. However, an
important factor that has been relatively unexplored in the
literature is how the choice of synthetic precursors inﬂuences the properties of these materials. Alkoxides, such as
tetraethoxysilane (TEOS) and triethylphosphate (TEP), are
the most commonly used precursors for provide the system
with SiO2 and P2O5, respectively. Additionally, nitrates are
the commonly used sources of CaO and other metal oxides
in this system [3, 7, 18–25, 28–33].
Pereira et al.  demonstrated that, by exclusively
using alkoxide precursors in the synthesis of SiO2–CaO–
P2O5 gel-glasses, it is possible to obtain more homogeneous
calcium distributions in these materials than in others, such
as gel-glasses prepared using calcium nitrate as a precursor.
Ramila et al.  corroborated these results by preparing a
new gel-glass composition in the same system. This study
demonstrated that the exclusive use of alkoxides as synthetic precursors leads to more homogeneous glasses
because the most critical step in the nitrate-based methods,
i.e., the decomposition during the thermal-conversion of
gels into ceramics, does not occur. These authors also
showed through in vitro bioactivity tests that the growth of
an HA surface layer on this material occurs more uniformly
than on the material synthesised with calcium nitrate .
Even after these results, the use of calcium alkoxides in the
synthesis of bioactive gel-glasses and glass–ceramics is still
uncommon because these precursors have high hydrolysis
rates, which require special synthesis conditions [27, 36,
37]. In contrast, calcium nitrate is easy to handle, is very
soluble in the reaction medium, and also yields highly
bioactive materials [3, 7, 18–25, 28–34, 37].
Further investigations of the inﬂuence of precursors
used in synthesis on the properties of these materials can
also be found in the published works of Łaczka et al. [15,
17], in which variations in the precursors led to the formation of different crystalline phases under the same heat
treatment conditions; additionally, the crystallisation temperatures (Tc) of the studied systems varied signiﬁcantly.
Thus, not only is the ﬁnal composition reﬂected in the
properties of the bioactive glasses and glass–ceramics
prepared by sol–gel processing but also the nature of the
precursors employed in their synthesis. In this context, the
present work discusses the synthesis, characterisation and
in vitro bioactivity properties of gel-glasses and glass–
ceramics of the SiO2–CaO–P2O5 system prepared with
different phosphorus precursors, which are selected based
on their respective rates of hydrolysis.
2 Materials and methods
2.1 Preparation of the gels
The nominal compositions established for the synthesis of
all samples of the SiO2–CaO–P2O5 system were
70:26:4 mol% of SiO2:CaO:P2O5, respectively. We chose
this system and the relative composition based on the initial
published studies related to the synthesis of bioactive gelglasses . Furthermore, the crystallisation behaviour of
bioactive gel-glasses with this composition, and similar
compositions, has been reported in the literature [18–20,
38]. Preparation of the gels involved hydrolysis and polycondensation reactions of stoichiometric amounts of TEOS
(Si(OC2H5)4; Aldrich), TEP (OP(OC2H5)3; Merck), and
calcium nitrate (Ca(NO3)2Á4H2O; Labsynth), as given by
the desired nominal composition stated above. The
hydrolysis of TEOS and TEP was catalysed by a solution
of 0.1 mol L-1 HNO3 using an 8:1 molar ratio of
HNO3 ? H2O to TEOS ? TEP. Beginning with the
hydrolysis of TEOS, the other reagents were sequentially
added to the reaction mixture in 60-min intervals while the
mixture was maintained under constant stirring. Before
reaching the gel point, the sols were poured into TeﬂonÒ
3. J Mater Sci: Mater Med
tubes and stored for 3 days. At the end of this period, the
gels were dried for 7 days at 70 °C and 2 days at 130 °C.
After completion of the drying step, the gels were crushed
manually in an agate mortar, and the powders were
selected according to particle size (150 lm) and then
characterised. All samples prepared from the TEP precursor were identiﬁed as Bio1_TEP.
The same synthesis procedure described for TEP preparation was also employed for the preparation of gels from
the other phosphorus precursors. The second set of samples
was prepared from phosphoric acid (H3PO4; Qhemis) and
was identiﬁed as Bio2_AFos. A criterion was established
for the choice of the last phosphorus precursor and subsequent gel preparation. Previous studies [27, 39–41] have
shown that phosphorus precursors with the structure
OP(OH)3-x(OR)x exhibit intermediate chemical reactivity
to compounds of type OP(OR)3 and OP(OH)3. Thus, a
precursor with the OP(OH)3-x(OR)x structure was chosen
because TEP and H3PO4 have the structures OP(OR)3 and
OP(OH)3, respectively. The OP(OH)3-x(OR)x precursor
was obtained by dissolving phosphorus oxide (P2O5; J.T.
Baker) in ethanol according to the stoichiometry of the
simpliﬁed chemical reaction below:
P2 O5 þ 3EtOH ! OPðOHÞ2 ðOEtÞ þ OPðOHÞðOEtÞ2
where Et represents –CH2CH3. The methods used to prepare and analyse this precursor are given in the supplementary material. All samples resulting from the use of this
precursor were identiﬁed as Bio3_EFos.
Table 1 Heat treatment program for converting the Bio1_TEP gels
into glasses and glass–ceramics
Samples derived from the Bio2_AFos and Bio3_EFos gels underwent the same heat treatments
atmosphere (air). The heating program was determined
based on the analytical results of previous differential
scanning calorimetry (DSC) and thermogravimetry (TG)
analyses. The heat treatment consisted of heating at
5 °C min-1, followed by an isothermal hold at a temperature selected according to the data shown in Table 1. The
samples were allowed to cool naturally in the electric
After the heat treatments had been carried out, the
resulting powders were manually crushed in an agate
mortar, and powders with particle sizes of 25–75 lm were
selected and characterised. The ﬂowchart in Fig. 1 outlines
the procedures established for particulate gel preparations,
which were later characterised and then subjected to thermal treatments to obtain the bioactive glasses and glass–
ceramics of the SiO2–CaO–P2O5 system.
2.2 Conversion of the gels into glasses and glass–
2.2.1 In vitro bioactivity tests
After milling, gels with particle sizes smaller than 150 lm
were selected, and individual portions containing approximately 20 g were placed in ZAS (ceramic material based
on zirconia, alumina and silica) crucibles for heat treatment. The gel particles were heat-treated in an electric
furnace at high temperatures under an oxidising
To evaluate the bioactivity of the synthesised materials, we
performed in vitro tests according to the method described
by Kokubo and Takadama . The solution employed in
these tests is known as simulated body ﬂuid (SBF); SBF is
acellular, protein-free and has a pH of 7.40. Additionally,
its ionic concentration versus that of human blood plasma
Fig. 1 Flowchart of the steps involved in preparing the particulate gels and their subsequent conversion into glasses and glass–ceramics
4. J Mater Sci: Mater Med
can be seen in the supplementary material. This solution is
often used and indicated for in vitro evaluations of the
formation of HA surface layers on materials designed for
implants, according to ISO 23317 .
2.2.2 Preparation of the samples
To test the in vitro bioactivity, the previously characterised
powders with particle sizes of 25–75 lm were reformed into
pellets measuring 10 mm in diameter and 2.3 mm in height.
The reforming process consisted of two steps. First, the
powders were uniaxially pressed at 65 MPa for 6 min with no
agglutinant. The second stage was performed in an isostatic
press at 170 MPa for 3 min. Each pellet was ﬁrst tied by
nylon around its circumference and then cleaned ultrasonically for 15 s in acetone. Following cleaning, pellets were
dried and then soaked in PET bottles containing the SBF.
The volume of SBF used in the bioactivity tests is
determined as a function of the sample surface area.
According to the procedures described by Kokubo and
Takadama  for a dense material, the appropriate volume of solution obeys the following relationship:
where Vs represents the volume of SBF (mL) and Sa, the
total geometric area of the sample (mm2). For porous
materials, such as the pressed pellets used in this study, the
authors also suggest the use of a volume in excess of that
calculated by Eq. (1) Therefore, in this study, we used
the following procedure: sample mass (m) divided by the
volume of SBF (Vs) equal to 0.01 g mL-1 because all the
pellets had the same m. During the tests, the pellets were in
contact with the SBF for periods of 3, 6, 12, 24, 48, 72, 96,
120 and 144 h, and the system temperature was held at
37 °C using the heating device illustrated in Fig. 2. Following the predetermined test time, the pellets were taken
out of their bottles and immersed in acetone for 10 s to
remove the solution and to halt any surface reaction.
Shortly after drying, both pellet surfaces were inspected for
the formation of a HA surface layer.
Fig. 2 Schematic
representation of the procedures
adopted for performing the
in vitro bioactivity tests
2.2.3 Evaluation of solubility of the samples in SBF
To evaluate the solubility of the samples in the SBF during
the bioactivity tests, the ionic concentrations of H?, Ca2?
and P-PO43- were analysed an average of three times per
testing time. This procedure enabled the identiﬁcation of the
dissolution behaviour of the samples during the tests. The
concentrations of H? and Ca2? were measured according to
the ion-selective electrode technique using a Roche cobas b
121 electrolyte analyser system. Ultraviolet and visible
spectrophotometry (UV–Vis) were used for the P-PO43concentration measurements because aqueous inorganic
phosphate ions react with certain compounds to form a blue
chromophore whose colour intensity is proportional to its
concentration in the medium (see more details in supplementary material). These measurements were taken with a
Siemens ADVIAÒ 1800 clinical analyser.
2.3 Characterisation of the materials
2.3.1 Differential scanning calorimetry
and thermogravimetry (DSC/TG)
DSC and TG analyses were performed in a Netzsch STA
449 C instrument under an oxidising atmosphere (synthetic
air with a gas ﬂow of 50 mL min-1). Typical analyses
involved gel particle samples weighing approximately
30 mg and a heating program of 5 °C min-1 from room
temperature to 1,200 °C to determine the initial heat
treatment temperature and the onset of crystalline phase
precipitation in the material.
2.3.2 X-ray diffraction (XRD)
The glassy materials and crystalline phases that resulted
from the heat treatments of the gels were characterised by
XRD using a Siemens model D5000 diffractometer operating with CuKa radiation (k = 0.15418 nm). The diffraction patterns were obtained in the 2h range from 10° to
70° in a continuous scan mode at 2° min-1.
5. J Mater Sci: Mater Med
Fig. 3 Illustration of the Bio1_TEP (a), Bio2_AFos (b) and Bio3_EFos (c) gels synthesised with the use of TEP, phosphoric acid and a
phosphorus precursor previously prepared from dissolving P2O5 in ethanol (24 h reﬂux), respectively
2.3.3 Fourier transform infrared spectroscopy (FTIR)
The presence of pellet surface modiﬁcations after the
in vitro bioactivity tests was assessed by FTIR using a
Perkin Elmer Spectrum GX spectrometer operating in
reﬂectance mode with a 4 cm-1 resolution in the
400–4,000 cm-1 region. FTIR spectra were also obtained
in transmission mode using the KBr pellet technique,
which, together with the liquid-state nuclear magnetic
resonance of phosphorus-31 (31P-NMR), allowed for conﬁrmation of the formation of a OP(OH)3-x(OEt)x species
following ﬁxed reaction times with P2O5 and ethanol.
P-NMR spectra were obtained using a Bruker DRX 400
spectrometer at 9.4 Tesla and 162 MHz. Phosphoric acid
(H3PO4 85 %) was used as a reference. The results and
discussion of the FTIR and 31P-NMR spectra of these
solutions can be accessed in the supplementary material.
2.3.4 Scanning electron microscopy and microanalysis
The pellets were morphologically characterised by SEM to
determine the surface modiﬁcations that occurred during
the in vitro bioactivity tests. A set of samples was selected
and analysed before and after soaking in the SBF for different testing times. The samples were coated with an
evaporated gold ﬁlm to render the surface electroconductive; then, the samples were analysed under a Phillips FEG
X-L30 microscope coupled with an energy dispersive
X-ray spectrometer (EDS), which allowed for qualitative
chemical analysis of the sample surfaces.
3 Results and discussion
3.1 Synthesis and characterisation of the Bio1_TEP
The time required for the reaction mixture to become relatively rigid and cease ﬂowing was considered to be the
Fig. 4 DSC and TG curves of the Bio1_TEP gel
gelation time. The gels synthesised using TEP as the phosphorus precursor presented a gelation time of approximately
70 h. After drying, these gels were transparent, colourless
and optically homogeneous, as shown in Fig. 3a.
Simultaneous DSC and TG analyses were performed
with a fraction of the Bio1_TEP gel particles to determine
the heat treatment program. The results of these analyses
are shown in Fig. 4, and they agree well with other
reported studies [3, 15, 18, 21, 25]. The gel underwent
three distinct mass loss steps before becoming effectively
stable at approximately 600 °C. The ﬁrst mass loss stage
occurs at approximately 130 °C and can be associated with
the endothermic desorption of the physically adsorbed
water. At approximately 280 °C, the volatilisation of water
is also observed, although here it is much less pronounced;
additionally, at this temperature desorption is an exothermic chemical process. Between 300 and 550 °C, the mass
loss steps were more pronounced. The highly endothermic
ﬁrst process, which is centred at 370 °C, is attributed to the
evolution of incomplete condensation products of the precursors, such as the alkoxide groups. The second process in
this region is the most critical, in which the maximum mass
ﬂow occurred from the solid to the vapour phase at 451 °C.
6. J Mater Sci: Mater Med
This mass loss step corresponds to the removal of nitrate
ions from the material by means of their decomposition.
The exothermic ﬁnal process occurs at approximately
855 °C and is related to the onset of crystallisation.
Based on the DSC and TG analyses, the initial heat
treatment temperature was set at 700 °C and maintained for
3 h, which should be a sufﬁcient condition for the complete
elimination of nitrate ions and the formation of a glassy
material because crystallisation is only observed near
855 °C. Conﬁrming this hypothesis, in the X-ray diffractogram of the Bio1(1)_TEP sample after this treatment it is
only possible to observe an amorphous halo at approximately
25° (2h); such a feature is typical of glassy silicate materials.
After establishing the conditions necessary to achieve
glass formation, the second step was to determine the
temperature at which crystallisation is initiated toward the
design of other thermal treatments and subsequent glass–
ceramic preparations. A DSC run was performed for the
Bio1(1)_TEP glass, and the result is shown in Fig. 5. First,
the endothermic peak located at 64 °C is associated with
the volatilisation of physically adsorbed water in the
material. Then, the exothermic peaks at 855, 955 and
1074 °C are attributed to crystallisation. Another exothermic process can be observed at approximately 1,180 °C but
is much less pronounced than the others. It is important to
mention that the powders tested in Fig. 5 are largely glassy
because they show quite prominent crystallization peaks in
the DSC traces. Therefore, we guess the small baseline
deviation normally associated to Tg was hidden in the
background of the rather noisy DSC curves.
The onset of the Bio1(1)_TEP glass crystallisation
process is indicated on the graph by Tc (crystallisation
temperature); this temperature was identiﬁed by locating
Fig. 5 DSC curves of the Bio1(1)_TEP, Bio2(1)_AFos and
Bio3(1)_EFos glass samples
the intersection of a line that extends beyond the baseline
with the curve tangent at the inﬂection point of the ﬁrst
exothermic peak, which is located at 855 °C. The calculated value (intersection point) was 829 °C. Based on these
data, the thermal treatments needed to obtain the glass–
ceramics were determined to be 900, 1000, 1100 and
1200 °C. Figure 6 shows the XRD patterns that were
obtained from the Bio1_TEP samples that were submitted
to these heat treatments.
Based on inspections of the XRD patterns, evidence of
crystallisation can only be found for the heat treatments
above 800 °C, which is consistent with the DSC data
acquired for the Bio1(1)_TEP glass. At 900 °C, the primary identiﬁed phases were apatite (Ca5(PO4)3(OH)) and,
signiﬁcantly less pronounced, wollastonite (CaSiO3). At
1,000 °C, these phases, especially the wollastonite, became
more pronounced, and a new phase, quartz (a-SiO2),
emerged. The results obtained at 1,100 °C were very
similar to those obtained from the heat treatment carried
out at 1,000 °C with the only noticeable alteration
observed as a decrease in the quartz peak intensities. In the
ﬁnal heat treatment (1,200 °C), the decreased intensities of
the quartz peaks were maintained; additionally, two more
phases were formed: tricalcium phosphate (a-Ca3(PO4)2)
and cristobalite, which is an a-SiO2 polymorph. It should
be noted that even the Bio1(6)_TEP samples thermally
treated at 1,200 °C exhibited a residual glassy phase, which
may be veriﬁed by the presence of a low-intensity
Fig. 6 XRD patterns of the samples derived from the Bio1_TEP gel:
ﬁlled square apatite, ﬁlled circle wollastonite, ﬁlled triangle quartz,
open circle tricalcium phosphate, open square = cristobalite
7. J Mater Sci: Mater Med
amorphous halo centred at approximately 25° (2h). This
agrees well with the results described by Padilla et al. 
in a study performed with similar materials.
3.2 Synthesis and characterisation of the Bio2_AFos
The gels synthesised with phosphoric acid showed a
gelation time of approximately 50 h. The appearance of
the gels obtained after drying was similar to that of the
Bio1_TEP gels, as shown in Fig. 3b. After preparing the
gel particles, the same previously established heat treatment program was employed, and the XRD results of these
samples are shown in Fig. 7.
The XRD patterns of the samples treated at 700 and
800 °C were typical of glassy materials; however, the XRD
pattern of the Bio2(2)_AFos sample, which was heattreated at 800 °C, had a broad peak centred at approximately 32° (2h). This peak is attributed to the presence of
apatite, whose intensity is not strong enough to establish
the extent of the sorting feature for this phase [3, 19, 20].
At 900 °C, the presence of the apatite phase became much
more pronounced, and wollastonite and tricalcium phosphate had formed. At 1,000 and 1,100 °C, a substantial
increase in the fractions of those phases was found, and
discrete peaks associated with quartz appeared in the XRD
patterns of the sample heat treated at 1,100 °C. At
1,200 °C, the cristobalite phase formed and there was an
Fig. 7 XRD patterns of the samples derived from the Bio2_AFos gel:
ﬁlled square apatite, ﬁlled circle wollastonite, ﬁlled triangle quartz,
open circle tricalcium phosphate, open square = cristobalite
upward trend in the intensities of the peaks of the phases
already present, indicating their further development.
These results were quite different from those obtained
from the crystallisation of the Bio1_TEP samples, in which
the formation of quartz could be detected at lower temperatures and with greater intensity. Moreover, tricalcium
phosphate was observed only after these samples were
thermally treated at 1,200 °C (see Fig. 6). On the other
hand, the presence of glassy phase remnants following
complete heat treatment was identiﬁed in both sets of
samples, as shown in the diffractograms of Figs. 6 and 7 as
the presence of an amorphous halo centred at approximately 25° (2h).
3.3 Synthesis and characterisation of the Bio3_EFos
After conﬁrming the formation of the OP(OH)3-x(OEt)x
species, we then performed gel synthesis from the solution
obtained following 24 h of reﬂux time because the only
signiﬁcant difference between the samples was the existing
condensed phosphate concentration (see supplementary
material). According to Ali et al. , an increase in the
condensed phosphate concentration is not a drawback of
the sol–gel synthesis technique because these compounds
are able to hydrolyse and polycondense during the process.
Furthermore, the acidic characters of these species can
contribute to the initial hydrolysis of the monomeric precursors involved in the synthesis, which promotes a
mechanism similar to that observed in acid catalysis. When
performing synthesis from this precursor, it was conﬁrmed
that shorter gelation times were observed, with gel formation occurring within approximately 45 h. The gels
obtained after drying were optically homogeneous, transparent and slightly yellow, as shown in Fig. 3c.
The XRD patterns of the samples derived from the
Bio3_EFos gels following full heat treatment are shown in
Fig. 8. These diffractograms are similar to those obtained
for the samples derived from the Bio1_TEP gels that
converted into glass–ceramics, which are shown in Fig. 6.
For each heat treatment, the resulting phases were identical
between these two sample sets, with the only differences
restricted to differences in relative peak intensities as
pertaining to the phases present, especially those that are
associated with wollastonite, quartz and cristobalite.
Unlike that of the Bio1_TEP samples, the quartz phase that
formed in the Bio3_EFos samples evolved when the temperature was increased from 1,000 to 1,100 °C. A signiﬁcant reduction in the intensity of the quartz peaks was only
found at 1,200 °C, which may be related to formation of
the cristobalite. This phase is identiﬁed here by the
appearance of a poorly deﬁned cristobalite peak. With
8. J Mater Sci: Mater Med
Bio1_TEP gels, in which it was possible to observe a more
readily apparent intensity increase for such peaks.
3.4 In vitro bioactivity tests
Fig. 8 XRD patterns of the samples derived from the Bio3_EFos gel:
ﬁlled square apatite, ﬁlled circle wollastonite, ﬁlled triangle quartz,
open circle tricalcium phosphate, open square = cristobalite
regard to the wollastonite peaks, the increase in the heat
treatment temperature did not signiﬁcantly inﬂuence their
development as it did with samples derived from the
The morphologies of the some samples prepared from the
Bio1_TEP, Bio2_AFos and Bio3_EFos gels and the outcomes of their respective qualitative chemical analyses
before and after exposure to SBF at different testing times
are shown in Figs. 9 and 10. With only 3 h of testing, it was
possible to observe the formation of a surface layer of semispherical particles. The EDS analysis indicated that the
surface layer compositions of the pellets from this testing
time (3 h) were similar to the surface compositions of
samples without exposure to the SBF, aside from a noticeable increase in the concentrations of Ca and P versus the Si
concentration. This can be attributed to an increase in the
migration of these species to the material surface. After 24 h
of testing, all glass samples had morphologies typical of HA,
as shown in Figs. 9c–d and 10b [18, 19, 21, 22, 42, 44]. For
these morphologies, the formation of HA was already well
established and the HA grew increasingly dense with
increased testing times. By 144 h, it was no longer possible
to observe the presence of Si on the sample surfaces because
the surfaces were completely covered by the HA layer. All of
these developments are accompanied by corresponding EDS
spectra (see Figs. 9, 10).
Fig. 9 SEM micrographs and EDS spectra of the Bio3(1)_EFos sample surfaces: a before the test, b after 3 h, c after 24 h and d after 144 h
9. J Mater Sci: Mater Med
Fig. 10 SEM micrographs and EDS spectra of the Bio1(1)_TEP and Bio2(6)_AFos samples surfaces: a and c before the test; b and d after 144 h
For all glass–ceramic samples, it was also possible to
observe the typical morphology of HA, as shown in
Fig. 10d. As observed in the EDS spectra of the
Bio2(6)_AFos samples (Fig. 10c–d), there is a large compositional change of the surfaces after 144 h of testing; the
surface compositions are characterised by a predominance
of Ca and P, which further veriﬁes the HA layer formation.
The initial formation of the HA layer on the surface of
these samples, as well as on the other glass–ceramic
sample surfaces, was not observed in this study because the
bioactivity tests were only carried out for the time of 144 h
to verify the in vitro bioactivity of these materials.
According to the SEM micrographs and EDS spectra,
evidence of HA layer formation on the surface of the glass
samples was only identiﬁed after exposure to the SBF for
over 24 h. The FTIR spectra conﬁrmed the formation of HA
on the surfaces of these samples at this time, as shown in
Fig. 11. From the spectra of the SBF-soaked samples, within
3 h of testing the initiation of the formation of a silica-rich
layer on the surface can be observed. This observation is
further evidenced by deformation of the Si–O–Si associated
bands, which are located at approximately 1,115 and
1,255 cm-1 . In the same testing period, a broad band
appears at approximately 575 cm-1; this band is associated
with the vibrational mode of dP–O bonds, which are caused
by the growth of amorphous calcium phosphate in the region
where silica-rich layer had initially formed [5, 21]. This band
becomes more deﬁned with increasing reaction time and is
subsequently divided into two vibrational modes near 565
and 605 cm-1, which are both characteristic of HA [3, 5, 7,
18, 21, 22, 34, 36, 37, 44]. Additionally, the emergence of
two new bands associated with the formation of HA can be
observed in the spectrum. These bands are associated with
the vibrational modes of the P–O and P=O bonds at
approximately 1,055 and 1,130 cm-1, respectively. After
24 h of testing, the collected FTIR spectra are similar to the
synthetic HA spectrum, with further changes related only to
the intensities of the bands as a function of testing time [3, 5,
7, 18, 21, 22, 34, 36, 37, 44]. These changes indicate greater
HA density on the sample surfaces with advancing stages of
The Bio1(1)_TEP, Bio2(1)_AFos and Bio3(1)_EFos
glass samples obtained from the thermal treatment at
700 °C responded similarly to in vitro bioactivity tests.
Their spectra differed only from the spectra acquired
before the tests, as shown in Fig. 11. The Bio1(1)_TEP and
Bio3(1)_EFos glass spectra acquired before the tests were
almost identical; meanwhile, the Bio2(1)_AFos glass
spectrum showed additional bands at approximately 570
and 600 cm-1, which were associated with the stretching
of phosphate group bonds . These results suggest the
possible segregation of PO43- groups in the glass structure
that was prepared from the Bio2_AFos gels synthesised
using phosphoric acid.
The FTIR spectra of the glass and glass–ceramic samples obtained from thermal treatments above 700 °C, both
10. J Mater Sci: Mater Med
Fig. 11 FTIR spectra of the glass sample surfaces before and after soaking in SBF for different testing times
Fig. 12 FTIR spectra of the glass and glass–ceramic sample surfaces before and after 144 h of soaking in SBF
11. J Mater Sci: Mater Med
Fig. 13 Variation in the pH and the calcium and phosphorus concentrations as functions of the exposure time of the glass samples to SBF
before and after exposure to the SBF for 144 h, are shown
in Fig. 12. From these spectra, it can observed that all
samples exhibit an in vitro bioactive behaviour, which is
identiﬁable by the bands located at approximately 565,
605, 1055 and 1130 cm-1 and attributed to the formation
of a HA layer [3, 5, 7, 18, 21, 22, 34, 36, 37, 44]. Low
intensity bands associated with quartz (approximately 780
and 800 cm-1) and wollastonite (approximately 640, 680,
720, 900, 940 and 1050 cm-1) could also be identiﬁed in
some samples without exposure to the SBF [18, 19].
The variations in pH and in the calcium and phosphorus
concentrations as a function of the exposure time to the
SBF for the Bio1(1)_TEP, Bio2(1)_AFos and Bio3(1)_
EFos samples is presented in Fig. 13. This procedure
serves as a parameter to assess the in vitro bioactivity of the
According to our analysis, the pH increases from 7.4 to 7.8
within the ﬁrst 24 h of testing. This rise in pH is directly
related to the Ca2? concentration in solution, which increases following rapid dissolution of the material; in this
time period, the concentration of Ca2? changed by
2.9 mmol L-1. It is worth mentioning that the pH increases
as a function of ion exchange between the Ca2?, appeared
due to the material dissolution in the medium, and H?,
already present in solution. After 24 h of testing, a ﬂuctuation in the concentration of Ca2? can be observed with a
globally decreasing trend, except for the Bio2(1)_AFos
samples. In terms of P-PO43-, their concentration in solution
falls considerably after 3 h and is almost depleted at testing
times in excess of 72 h. This consumption is due to the early
formation of an amorphous calcium phosphate layer on the
surfaces of the materials and its subsequent evolution into
HA (see FTIR spectra in Fig. 11). These steps depend almost
exclusively on the phosphorus provided by the solution
because at the appointed testing times could not be detected
any P-PO43- increase in the SBF. Although all sets of samples showed similar behaviour in this test, it is notable that
the Bio2(1)_AFos samples showed the highest solubility,
indicating that the segregation of PO43- units in the glass had
no adverse effect on the bioactivity of this material.
In Fig. 14, the pH and the concentrations of calcium and
phosphorus after 144 h of exposure of all glass and glass–
ceramic samples to the SBF are compared as a function of
the heat treatment temperature. There is a decrease in the
reactivity of all materials with increasing treatment temperature; this trend is especially pronounced above 900 °C.
It should be noted that from this point all samples already
exhibited the presence of crystalline phases. With the
introduction of crystallinity in these materials, the bioactivity is governed by the crystalline phases present, in
addition to their crystallised fractions. This can be clearly
observed in a comparison of the samples derived from
the Bio1_TEP and Bio3_EFos gels, whose X-ray diffractograms show very similar crystallisation behaviours, with
the only difference being the peak intensities of each phase
formed after heat treatment. In this case, the reactivity of
these samples was distinct when they were compared again
after soaking in the SBF for 144 h.
12. J Mater Sci: Mater Med
Fig. 14 Variation in the pH and the calcium and phosphorus concentrations after 144 h of exposure of the glass and glass–ceramic samples to
In XRD patterns, the relative intensities of the peaks of
each crystalline phase are proportional to its existing
fraction, which allowed the importance of the wollastonite
phase in the bioactivity of these materials to be interpreted.
The same relevance to bioactivity could not be observed
for the apatite. For the Bio3_EFos glass–ceramic samples
(obtained above 900 °C), that the formation and evolution
of the wollastonite was not signiﬁcantly favoured with heat
treatments, which was in contrast to apatite, almost no
variation was observed in the Ca2? concentration in the
SBF. Hence, no larger changes in the pH of the medium
were observed. The variation in the P-PO43- concentration
was also not signiﬁcant at an approximate value of 0.56
versus 0.71 mmol L-1, which was observed for the
Bio1_TEP glass–ceramic samples. These values are related
to the reduction of the P-PO43- concentration in solution;
therefore, comparing them with the value obtained for the
glass samples (approximately 0.95 mmol L-1), it is reasonable to say that the time required for HA layer formation on the material surface is the lowest for the glass
samples, followed by the Bio1_TEP and Bio3_EFos glass–
ceramic samples, respectively.
For the Bio2_AFos glass–ceramics, the reactivities were
higher than those of the Bio1_TEP and Bio3_EFos samples, as observed in Fig. 14, which are presented as functions of the higher activities of H?, Ca2? and P-PO43-.
This may be associated with the lower degree of crystallinity of these samples and with the presence of wollastonite and tricalcium phosphate, which both exist in these
samples following thermal treatments performed at 900 °C
and above. It is worth reiterating that the tricalcium
phosphate phase was detected in the Bio1_TEP and
Bio3_EFos samples only after the completion of heat
treatments at 1,200 °C, as demonstrated in the diffractograms presented in Figs. 6 and 8, respectively. The most
stable materials in the SBF during the in vitro bioactivity
tests were those obtained after heat treatments at 1,100 and
1,200 °C. This high stability is attributed to the quartz and
cristobalite phases, which were present in all sets of samples subjected to these treatments. In the case of cristobalite, this was only true for the samples subjected to heat
treatment at 1,200 °C because this phase was only identiﬁed after thermal treatments carried out at this temperature.
4 Inﬂuence of phosphorus precursors on the synthesis
By simply varying the phosphorus precursor used in the
synthesis of the bioactive glasses and glass–ceramics with
compositions in the SiO2–CaOP2O5 system, signiﬁcant
changes were observed in the resulting materials, beginning as early as the time required for gel formation. The
longest gelation time, which was approximately 70 h, was
observed for gels prepared with TEP. The hydrolysis rate
of this precursor is considered the slowest versus the other
phosphorus precursors, which may explain this result. The
shortest gelation time was expected for gels prepared with
phosphoric acid; however, the shortest gelation time was
actually found in the samples prepared with the precursor
13. J Mater Sci: Mater Med
Fig. 15 Mineralisation behaviour of the Bio1_TEP, Bio2_AFos and Bio3_EFos gels as a function of the heat treatment temperature
generated by dissolving P2O5 in ethanol. The gels formed
from this precursor gelled within approximately 45 h,
which may be associated with the presence of the condensed phosphates that were identiﬁed by 31P-NMR (see
supplementary material). The condensed phosphates have
an acidic character and can potentially contribute to the
initial hydrolysis reactions of the system. For the gels
prepared with phosphoric acid, a gelation time of approximately 50 h was observed.
The main inﬂuence of the phosphorous precursors used
was undoubtedly on the conversion of the gels into glass–
ceramic materials. This is evidenced by the graph in
Fig. 15, in which the mineralisation behaviours of the
Bio1_TEP, Bio2_AFos and Bio3_EFos gels by heat treatment temperature are qualitatively demonstrated. It is
important to clarify that the graphs shown in Fig. 15 were
constructed by monitoring the most intense peak of each
crystalline phase identiﬁed in the X-ray diffractograms,
which are displaced in Figs. 6, 7 and 8. Because the relative intensities of these peaks are directly related to the
fractions of the existing phases, it was possible to monitor
the fractional increase or decrease of these phases in the
material as a function of the heat treatment temperature.
Therefore, an intensity equal to zero corresponds to an
absence of the crystalline phase in the material.
For the Bio2_AFos samples, which were prepared with
phosphoric acid, we observed a more differentiated mineralisation behaviour than for the other samples. In the
former samples, the crystallisation temperature of the
quartz was shifted to higher values, and the quartz peaks on
the diffractogram did not reach the maximum relative
intensity, as was observed for the Bio1_TEP and Bio3_
EFos samples. It is still possible to verify that the formation
temperatures of apatite and tricalcium phosphate are
signiﬁcantly lower in these samples, and thus that the
glass–ceramic compositions are quite different from the
Bio1_TEP and Bio3_EFos glass–ceramics compositions
prepared at the same temperatures. Comparing the Bio1_
TEP and Bio3_EFos sample sets, it can be concluded that
both sets exhibit highly similar behaviour with increasing
heat treatment temperatures. The most signiﬁcant difference between these sample sets was the propensity for the
co-evolution of apatite and wollastonite in the Bio1_TEP
samples. For Bio3_EFos samples, only the development of
apatite was favoured.
The DSC curves of the –Bio1(1)_TEP, Bio2(1)_AFos
and Bio3(1)_EFos glasses are shown in Fig. 5. The analysis of these curves reveals that the initial phases of the
crystallisation processes in these materials, which is indicated on the graph by the crystallisation temperature (Tc),
were very similar. The Tc values observed for the
Bio1(1)_TEP and Bio3(1)_EFos glasses were 829 and
827 °C, respectively. For the Bio2(1)_AFos glass, this
value was slightly lower (788 °C). This result can be
used to explain the small crystallised fraction in the
Bio2(2)_AFos glass obtained by heat treatment at 800 °C.
Interestingly, even the Bio2_AFos samples, which have a
reduced onset temperature of crystallisation, exhibited
greater resistance to devitriﬁcation. This can be veriﬁed by
both the shapes and intensities of the exothermic peaks in
14. J Mater Sci: Mater Med
the DSC curves, which were associated with the sample
crystallisation behaviour. This is also evidenced by the
decreased resolution and intensities of the peaks in the
X-ray diffractograms of these samples, as shown in Figs. 7
For the in vitro bioactivity tests, we demonstrated that
all synthesised materials were bioactive by identifying the
HA layer on their surfaces. In general, we observed a
downward trend in the reactivity of these materials as a
function of increasing heat treatment temperature. That is,
the crystallised materials were more stable in the SBF, and
in these cases, the bioactivity was dependent on the crystalline phases as well as on their respective fractions in the
material. This explains the different reactivities observed
for the crystallised Bio1_TEP, Bio2_AFos and Bio3_EFos
sample sets obtained under the same heat treatment conditions. For these samples, both the crystalline phases and
their respective fractions differed considerably, as reﬂected
in their different reactivities. In this way, the importance of
the wollastonite and tricalcium phosphate phases to the
overall bioactivity of the investigated materials was veriﬁed.
The apatite phase was also conducive toward increased
bioactivity, but to a lesser degree than either the wollastonite
or tricalcium phosphates. In contrast, the quartz and cristobalite phases were highly stable and therefore unfavourable
Finally, it is important to note that in 2010 we also
proposed the use of phytic acid (C6H18O24P6) for the
synthesis of these materials because phytic acid is nontoxic
and presents features of considerable chemical reactivity.
However, the synthesis conditions established in our initial
study were not suitable for this precursor. The gels synthesised were transparent, amber but not homogeneous;
amorphous calcium phosphate precipitated in the reaction
medium . Recently, in further support of this promising
area of study, Ailing and Dong  reported the use of
phytic acid as a phosphorus precursor in bioactive sol–gel
CaO–SiO2–P2O5 glass synthesis, demonstrating that the
phytic acid helped calcium ions to enter the gel network
without the need of further calcination treatments.
Bioactive glasses and glass–ceramics of the SiO2–CaO–
P2O5 system were synthesised by a sol–gel route using
different phosphorus precursors. The synthesis was successfully carried out employing TEP, phosphoric acid and a
precursor prepared by dissolving phosphorus oxide in
ethanol. For these three different syntheses, the substitution
of only the synthetic precursors was sufﬁcient to signiﬁcantly inﬂuence the resulting materials, especially in terms
of the mineralisation behaviour of the gels during the heat
All synthesised materials were bioactive, as demonstrated by the formation of a HA layer on their surfaces
during the in vitro tests. In general, the bioactivity of these
materials decreased with increasing heat treatment temperature, especially above 900 °C, by which point the
samples were already partially crystallised. For these
crystallised materials, the samples prepared using phosphoric acid exhibited the best performance. This was due to
the preferential formation of wollastonite and tricalcium
phosphate and the increased resistance to devitriﬁcation
exhibited by these samples following the application of
Acknowledgments We extend our appreciation to Dr. Aluısio
A. Cabral Junior, from the Instituto Federal de Educacao, Ciencia e
Tecnologia do Maranhao, Brazil, for the DSC and TG analyses and to
Dr. Olga S. Dymshits, from NITIOM State Optical Institute, Russian
Federation, for the valuable instructions and discussions. We also
thank the Brazilian funding agencies Fundacao de Amparo a Pesquisa
do Estado de Sao Paulo—FAPESP (2007/08179-9) and Coordenacao
de Aperfeicoamento de Pessoal de Nıvel Superior—CAPES for the
ﬁnancial support used to accomplish of this research project.
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