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Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5
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Group5.the influence of phosphorus precursors on the synthesis and bioactivity si o2 cao-p2o5

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  • 1. J Mater Sci: Mater Med DOI 10.1007/s10856-012-4797-x The influence of phosphorus precursors on the synthesis and 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 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 e-mail: rastosfix@gmail.com URL: lamav.weebly.com 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 phosphate (a-Ca3(PO4)2). 1 Introduction 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 [1]. 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. [3], which led to a patent in 1991 concerning the production 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 approximately 90 mol% SiO2. The properties observed in the SiO2–CaO–P2O5 sol–gel glasses are entirely opposite those 123
  • 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 [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 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. [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 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 123 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 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 [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 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 Samplea Final treatment temperature (°C) Duration (min) Bio1(1)_TEP 700 180 Bio1(2)_TEP 800 180 Bio1(3)_TEP Bio1(4)_TEP 900 1,000 180 180 Bio1(5)_TEP 1,100 180 Bio1(6)_TEP 1,200 180 a 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 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 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– ceramics 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 [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 Fig. 1 Flowchart of the steps involved in preparing the particulate gels and their subsequent conversion into glasses and glass–ceramics 123
  • 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 [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 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 [42] for a dense material, the appropriate volume of solution obeys the following relationship: Vs ¼ Sa 10 ð1Þ 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 123 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. 31 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 (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 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 samples 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. 123
  • 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 123 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. [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 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 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 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 123
  • 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 123
  • 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 [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 samples obtained from thermal treatments above 700 °C, both 123
  • 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 123
  • 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 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 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. 123
  • 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 SBF 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 123 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 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, 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 123
  • 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 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 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 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 synthesised 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 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 123 of the mineralisation behaviour of the gels during the heat treatments. 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 thermal treatments. ´ Acknowledgments We extend our appreciation to Dr. Aluısio ˆ ´ 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. References 1. Hench LL. The story of bioglass. J Mater Sci: Mater Med. 2006;17:967–78. 2. Hench LL. Genetic design of bioactive glass. J Eur Ceram Soc. 2009;29:1257–65. 3. Li R, Clark AE, Hench LL. An investigation of bioactive glass powders by sol–gel processing. J Appl Biomater. 1991;2:231–9. 4. Li R, Clark AE, Hench LL. Alkali-free bioactive sol–gel compositions. Patent International Publication Number WO1991/ 017965 (1991). 5. 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