The influence of phosphorus precursors on the synthesisand bioactivity of SiO2–CaO–P2O5 sol–gel glasses and glass–ceramics

Renato Luiz Siqueira • Edgar Dutra Zanotto

Received: 12 June 2012 / Accepted: 15 October 2012

� 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 diffrac-

tion, 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 treat-

ments 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 pre-

pared using the phosphoric acid, which had a reduced onset

temperature of crystallisation and an increased resistance to

devitrification. However, all resulting materials were bio-

active. The in vitro bioactivity of these materials was

strongly affected by the heat treatment temperature. In

general, their bioactivity decreased with increasing treat-

ment temperature. For crystallised samples obtained above

900 �C, the bioactivity was favoured by the presence of two

crystalline phases: wollastonite (CaSiO3) and tricalcium

phosphate (a-Ca3(PO4)2).

1 Introduction

Melt-quenched silicate glasses containing calcium, phos-

phorus 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 [1]. In addition to

their bioactivity, i.e., their ability to form in situ

hydroxyapatite (HA) layers on their surfaces, which pro-

motes 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 osteo-

blast 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.

[3], which led to a patent in 1991 concerning the produc-

tion of the first alkali-free bioactive glass compositions [4].

These authors demonstrated that the bioactive gel-glasses

of the ternary SiO2–CaO–P2O5 system exhibited in vitro

bioactivity, even for compositions containing approxi-

mately 90 mol% SiO2. The properties observed in the

SiO2–CaO–P2O5 sol–gel glasses are entirely opposite those

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10856-012-4797-x) contains supplementarymaterial, 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

e-mail: rastosfix@gmail.com

URL: lamav.weebly.com

R. L. Siqueira � 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

123

J Mater Sci: Mater Med

DOI 10.1007/s10856-012-4797-x

observed for glasses prepared by traditional melt-quench-

ing 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 intrin-

sically 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 tis-

sue-engineering applications [13, 14]. Additionally, it is

possible to significantly improve the mechanical perfor-

mance of these materials via their glass–ceramic transfor-

mations by means of a controlled crystallisation process

[15–20].

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 Bioglass� 45S5 and Biosilicate� also have been reported

[33–35]. These materials are of the same quaternary SiO2–

CaO–Na2O–P2O5 system and also show very high bioac-

tivity 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 influ-

ences 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. [36] 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. [37] corroborated these results by preparing a

new gel-glass composition in the same system. This study

demonstrated that the exclusive use of alkoxides as syn-

thetic 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 [37].

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 for-

mation of different crystalline phases under the same heat

treatment conditions; additionally, the crystallisation tem-

peratures (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 gel-

glasses [3]. 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 poly-

condensation reactions of stoichiometric amounts of TEOS

(Si(OC2H5)4; Aldrich), TEP (OP(OC2H5)3; Merck), and

calcium nitrate (Ca(NO3)2�4H2O; 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 Teflon�

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123

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 precur-

sor were identified as Bio1_TEP.

The same synthesis procedure described for TEP prep-

aration 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 sub-

sequent 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:

P2O5 þ 3EtOH! OP OHð Þ2 OEtð Þ þ OP OHð Þ OEtð Þ2where Et represents –CH2CH3. The methods used to pre-

pare and analyse this precursor are given in the supple-

mentary material. All samples resulting from the use of this

precursor were identified as Bio3_EFos.

2.2 Conversion of the gels into glasses and glass–

ceramics

After milling, gels with particle sizes smaller than 150 lm

were selected, and individual portions containing approx-

imately 20 g were placed in ZAS (ceramic material based

on zirconia, alumina and silica) crucibles for heat treat-

ment. The gel particles were heat-treated in an electric

furnace at high temperatures under an oxidising

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 temper-

ature selected according to the data shown in Table 1. The

samples were allowed to cool naturally in the electric

furnace.

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 ther-

mal treatments to obtain the bioactive glasses and glass–

ceramics of the SiO2–CaO–P2O5 system.

2.2.1 In vitro bioactivity tests

To evaluate the bioactivity of the synthesised materials, we

performed in vitro tests according to the method described

by Kokubo and Takadama [42]. 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

Table 1 Heat treatment program for converting the Bio1_TEP gels

into glasses and glass–ceramics

Samplea Final treatment

temperature (�C)

Duration

(min)

Bio1(1)_TEP 700 180

Bio1(2)_TEP 800 180

Bio1(3)_TEP 900 180

Bio1(4)_TEP 1,000 180

Bio1(5)_TEP 1,100 180

Bio1(6)_TEP 1,200 180

a Samples derived from the Bio2_AFos and Bio3_EFos gels under-

went the same heat treatments

Fig. 1 Flowchart of the steps involved in preparing the particulate gels and their subsequent conversion into glasses and glass–ceramics

J Mater Sci: Mater Med

123

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 [43].

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 ultrasoni-

cally 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 [42] for a dense material, the appropriate vol-

ume of solution obeys the following relationship:

Vs ¼Sa

10ð1Þ

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. Fol-

lowing 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.

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-PO43-

concentration 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 supple-

mentary material). These measurements were taken with a

Siemens ADVIA� 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 oper-

ating with CuKa radiation (k = 0.15418 nm). The dif-

fraction patterns were obtained in the 2h range from 10� to

70� in a continuous scan mode at 2� min-1.

Fig. 2 Schematic

representation of the procedures

adopted for performing the

in vitro bioactivity tests

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123

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 con-

firmation of the formation of a OP(OH)3-x(OEt)x species

following fixed reaction times with P2O5 and ethanol.31P-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

(SEM/EDS)

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 dif-

ferent testing times. The samples were coated with an

evaporated gold film to render the surface electroconduc-

tive; 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

samples

The time required for the reaction mixture to become rela-

tively rigid and cease flowing was considered to be the

gelation time. The gels synthesised using TEP as the phos-

phorus 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 exother-

mic 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 pre-

cursors, 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.

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

Fig. 4 DSC and TG curves of the Bio1_TEP gel

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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 diffracto-

gram 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 exother-

mic 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

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 calcu-

lated 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 pri-

mary 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. 5 DSC curves of the Bio1(1)_TEP, Bio2(1)_AFos and

Bio3(1)_EFos glass samples

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

J Mater Sci: Mater Med

123

amorphous halo centred at approximately 25� (2h). This

agrees well with the results described by Padilla et al. [19]

in a study performed with similar materials.

3.2 Synthesis and characterisation of the Bio2_AFos

samples

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 treat-

ment 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 heat-

treated at 800 �C, had a broad peak centred at approxi-

mately 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 phos-

phate 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

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 tem-

peratures 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 approxi-

mately 25� (2h).

3.3 Synthesis and characterisation of the Bio3_EFos

samples

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. [41], 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 pre-

cursors 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 for-

mation occurring within approximately 45 h. The gels

obtained after drying were optically homogeneous, trans-

parent 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 tem-

perature was increased from 1,000 to 1,100 �C. A signifi-

cant 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

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

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123

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

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

The morphologies of the some samples prepared from the

Bio1_TEP, Bio2_AFos and Bio3_EFos gels and the out-

comes 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 semi-

spherical 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 notice-

able 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. 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

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

J Mater Sci: Mater Med

123

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 com-

positional 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 [5]. 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

crystallisation.

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 [19]. 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 sam-

ples obtained from thermal treatments above 700 �C, both

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

J Mater Sci: Mater Med

123

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

J Mater Sci: Mater Med

123

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

tested materials.

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 increa-

ses 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 fluctua-

tion 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 sam-

ples 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 tem-

perature; 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 bioac-

tivity 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 diffrac-

tograms 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.

Fig. 13 Variation in the pH and the calcium and phosphorus concentrations as functions of the exposure time of the glass samples to SBF

J Mater Sci: Mater Med

123

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 rea-

sonable to say that the time required for HA layer forma-

tion 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 sam-

ples, as observed in Fig. 14, which are presented as func-

tions of the higher activities of H?, Ca2? and P-PO43-.

This may be associated with the lower degree of crystal-

linity of these samples and with the presence of wollas-

tonite 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 diffracto-

grams 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 sam-

ples subjected to these treatments. In the case of cristoba-

lite, this was only true for the samples subjected to heat

treatment at 1,200 �C because this phase was only identi-

fied after thermal treatments carried out at this temperature.

4 Influence of phosphorus precursors on the synthesis

and bioactivity

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, begin-

ning 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

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

SBF

J Mater Sci: Mater Med

123

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 con-

densed 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 approx-

imately 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 treat-

ment 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 rela-

tive 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 min-

eralisation 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 differ-

ence 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 anal-

ysis of these curves reveals that the initial phases of the

crystallisation processes in these materials, which is indi-

cated 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

Fig. 15 Mineralisation behaviour of the Bio1_TEP, Bio2_AFos and Bio3_EFos gels as a function of the heat treatment temperature

J Mater Sci: Mater Med

123

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

and 15.

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 crys-

talline 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 con-

ditions. 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 cristo-

balite phases were highly stable and therefore unfavourable

to bioactivity.

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 syn-

thesised were transparent, amber but not homogeneous;

amorphous calcium phosphate precipitated in the reaction

medium [45]. Recently, in further support of this promising

area of study, Ailing and Dong [46] 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.

5 Conclusions

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 suc-

cessfully 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 signifi-

cantly influence the resulting materials, especially in terms

of the mineralisation behaviour of the gels during the heat

treatments.

All synthesised materials were bioactive, as demon-

strated 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 tem-

perature, especially above 900 �C, by which point the

samples were already partially crystallised. For these

crystallised materials, the samples prepared using phos-

phoric 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

thermal treatments.

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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