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Supplementary material for the article “The influence of phosphorus precursors on the synthesis and bioactivity of SiO2–CaO–P2O5 sol-gel glasses and glass-ceramics”

Renato Luiz Siqueira (a, b) and Edgar Dutra Zanotto (b)

(a) Grupo de Pesquisas em Nanotecnologia e Nanomateriais, Centro Federal de Educação Tecnológica de Minas Gerais, Campus Timóteo, Av. Amazonas 1193, Vale Verde, 35183-006 Timóteo, MG, Brazil.

(b) Laboratório de Materiais Vítreos, Departamento de Engenharia de Materiais, Universidade Federal de São Carlos, Rod. Washington Luís km 235, CP 676, 13565-905 São Carlos, SP, Brazil.

Corresponding authors:

lamav.weebly.com

tel.: +55-16-3351-8556

fax: +55-16-3361-5404.

E-mail address: (Renato L. Siqueira)

1 Phosphorus OP(OH)3-x(OEt)x precursor

1.1 Synthesis

Using a method adapted from the literature [1-3], a stoichiometric amount of P2O5 was slowly added to 50 mL of ethanol under constant stirring. Because a highly exothermic reaction occurs, this process was conducted in an iced container to minimise losses through ethanol volatilisation. After complete dissolution of the P2O5, the resulting solution was refluxed at 78.5 °C (the boiling point of ethanol) for 12 and 24 h to eliminate any unreacted solvent fractions. Figure 1 shows the system used for the synthesis.

insert Figure 1 nearby (with ~90 mm width - supplementary material)

1.2 Characterisation

After refluxing for 12 and 24 h, the resulting solutions were highly viscous and slightly pink. The 31P-NMR and FTIR analyses are shown in Figure 2(a) and (b), respectively. Two groups of peaks can be seen in the 31P-NMR spectra. The first, near 0 ppm, was attributed to phosphoric acid (2.53 ppm), and mono- (1.29 ppm), di- (0.13 ppm) and tri-replaced (-0.73 ppm) OP(OH)3-x(OEt)x species, assuming x values of 0, 1, 2 and 3, respectively [1-4]. Based on the intensity of the peaks, the fractions of OP(OH)2(OEt) and OP(OH)(OEt)2 were very similar, while the amounts of phosphoric acid and triethylphosphate were minimal. The second group of peaks, located near 12 ppm, correspond to the condensed species of phosphate (Q1), consisting of trimers at -11.68 ppm and dimers at -12.65 ppm [1-4]. The concentration of these condensed phosphates in the solution increased significantly when the refluxing time was increased from 12 to 24 h, as can be observed by comparing their peak intensities in the spectra.

insert Figure 2 nearby (with ~90 mm width - supplementary material)

Significant differences in relation to solutions obtained with 12 and 24 h of refluxing were not observed in the FTIR spectra. The vibration modes exhibited in these spectra were characterised according to references [1-3]. At 3000 cm-1, a broad band corresponding to –OH stretches was observed. Other bands indicating the vibrational mode of the ν–CH3 bond were also observed in that region. Bands related to δ–CHx and ρ–CHx (x = 2 and 3) appeared at ~1440 cm-1 and between 1220-1080 cm-1, respectively. The area of larger absorption in the spectra was around 1050 cm-1, associated to the P–O and P=O stretching. In addition, P–O and O–P–O bonds signals were identified between 910 and 750 cm-1. Near 490 cm-1, another very intense band is seen in the spectra, and can be attributed to the vibrational mode of the P–O bond.

2 In vitro bioactivity tests

2.1 SBF ionic concentrations

insert Table 1 nearby (supplementary material)

2.2 Measurement of P-PO43- in the SBF by UV-Vis spectrophotometry

Monitoring of the P-PO43- concentration in the SBF after each testing time was based upon the reaction of phosphate ions with ammonium molybdate ((NH4)6Mo7O24·4H2O) in an acid medium to generate an ammonium phosphomolybdate complex ((NH4)3[P(Mo3O10)4]) [5]. This complex is reduced by sulphate-p-methylaminophenol ((CH3NHC6H4OH)2·H2SO4), resulting in a highly stable blue chromophore (heteropolymolybdate ([xMo12O40]n-)), whose absorbance at 660 nm is directly proportional to phosphate ions concentration in the analysed sample [6]. A simplified depiction of the sequence of reactions is given below:

phosphate ions + ammonium molybdate ammonium phosphomolybdate complex

ammonium phosphomolybdate complex heteropolymolybdate (chromophore)

Thus, the P-PO43- concentration was assessed as a function of each testing time by measuring the intensity of the absorbance of the heteropolymolybdate at 660 nm using a Siemens ADVIA® 1800 clinical analyser.

3 References

[1] B.I. Lee, W.D. Samuels, L-Q. Wang, G.J. Exarhos, Sol-gel synthesis of phosphate ceramic composites I, J. Mater. Res. 11 (1996) 134-143.

[2] A.F. Ali, P. Mustarelli, A. Magistris, Optimal synthesis of organo-phosphate precursors for sol-gel preparations, Mater. Res. Bull. 33 (1998) 697-710.

[3] D. Carta, D.M. Pickup, J.C. Knowles, M.E Smithc, R.J. Newporta, Sol-gel synthesis of the P2O5−CaO−Na2O−SiO2 system as a novel bioresorbable glass, J. Mater. Chem. 15 (2005) 2134-2140.

[4] J. Livage, P. Barboux, M.T. Vandenborre, C. Schmutz, F. Taulelle, Sol-gel synthesis of phosphates, J. Non-Cryst. Solids 147-148 (1992) 18-23

[5] C.H. Fiske, Y. Subbarow, The colorimetric determination of phosphorus, J. Biol. Chem. 66 (1925) 375-400.

[6] G. Gomori, A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter, J. Lab. Clin. Med. 21 (1942) 955-960.

[7] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity?, Biomaterials 27 (2006) 2907-2915.

Figure captions (supplementary material)

Figure 1. Schematic representation of the procedures adopted for performing the synthesis of the phosphorus OP(OH)3-x(OEt)x precursor from the reaction mixture containing P2O5 and ethanol.

Figure 2. 31P NMR (a) and FTIR (b) spectra of the solutions obtained from the reaction mixture containing P2O5 and ethanol.

Table captions (supplementary material)

Table 1. Ionic concentration of the human blood plasma and the SBF proposed for the evaluation of the in vitro bioactivity [7].