Chinese Chemical Letters  2016, Vol.27 Issue (04): 579-582   PDF    
Removal of residual nitrate ion from bioactive calcium silicate through soaking
Yong-Sen Suna, Ai-Ling Lia, Hui-Hui Rena,b, Xin-Ping Zhanga,b, Chao Wanga,b, Dong Qiua     
a Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China;
b University of Chinese Academy of Sciences, Beijing 100190, China
Abstract: Bioactive calcium silicates prepared by sol-gel routes mainly use calcium nitrate as the calcium precursor. However, the toxic nitrate ions are usually removed by calcination (i.e. 550℃ or over), which poses great challenge for the in situ preparation of inorganic/polymer composites, as polymer moieties could not survive such temperatures. In this study, we prepared 70Si30Ca (70 mol% SiO2 and 30 mol% CaO) bioactive glass at low temperatures where polymer could survive (i.e. 200℃ and 350℃), and proposed to remove the residual nitrate ions through soaking. Deionized water and simulated body fluid (SBF) were employed as the soaking medium. The results showed that the residual nitrate ions could be removed as quickly as 0.5 h while maintain the bioactivity of the samples. This technique may open the possibility of preparing sol-gel derived bioactive glass/polymer hybrids in situ with reduced potential toxicity.
Key words: Nitrate ion     Bioactive     Calcium silicate     Soaking     FTIR    
1. Introduction

Bone grafts, including autografts, allografts, xenografts and artificial materials are extensively used in clinics to replace or to repair bone tissues in defect sites resulted from disease or trauma [1]. The demand for artificial bone grafts has been raised as the supply of qualified natural bone grafts has diminished.

Bioactive glasses have showed excellent bioactivity thus have been increasingly used in dental and orthopedic surgeries to deal with bone-related diseases. 45S5, with its composition 45% SiO2, 24.5% CaO, 6% P2O5 and 24.5% Na2O (wt%), has been proven to bond both to bone or soft tissues through the formation of a bone-like hydroxycarbonate apatite (HCA) layer [2], thus becomes one of the most successful examples.

Although the melt-quenching-derived bioactive glasses have gained great commercial success, they are inherently brittle, which largely limits their application in weight bearing situations. Incorporation of polymeric moieties is expected to be a wise option. In order to get most use of both organic and inorganic moieties, a homogeneous mixing of them is a prerequisite. This can be in the form of either composite or hybrid materials. 45S5 is prepared through the melt-quenching method at high temperatures above 1700 ℃ [3, 4], which can hardly satisfy the above criteria. Therefore, a low-temperature sol-gel preparation method will be needed, to generate well-controllable micro/nanoparticles using as bioactive fillers or to form bioactive glass phase in the presence of polymer matrix.

A typical sol-gel procedure for bioactive glass involves the formation of a clear sol solution by the hydrolysis and condensation of silicon and calcium precursors, sol to gel transition, and the following aging, drying and stabilization stages. Tetraethyl orthosilicate (TEOS) or tetramethylorthosilicate (TMOS) usually serves as the precursor of silicon. And calcium nitrate usually serves as the precursor of calcium since it is highly soluble, low cost and more importantly, less thermal stable,i.e. relatively easier to be decomposed by heating compared with other inorganic calcium salts [5, 6, 7, 8, 9, 10]. Nevertheless, a minimal stabilization temperature over =0 ℃ is required to remove the potentially toxic nitrate ions [11, 12], which is well above the decomposition temperature of most biomedical polymers. For example, Koh et al. [13] prepared CaO-SiO2-PTMO hybrids at 40 ℃ only to find the existence of nitrate ions. The only strategy to fabricate in situ inorganic/ polymer hybrids is to lower the stabilization temperature to what polymer can survive. Although another calcium precursor, calcium 2-methoxyethoxide, has recently been explored with great success in preparing bioactive glass at lower temperature [14, 15], however it is not stable and often needs to be used freshly, thus still need more investigation. So far, the best choice for calcium is still calcium nitrate. Therefore, it is of great interest to explore new method to remove the residual nitrate ions in the bioactive glasses made from calcium nitrate.

Sol-gel-derived 70Si30Ca calcium silicate (70 mol% SiO2 and 30 mol% CaO) is a Class A biomaterial, which has great potential to be used as the third generation biomaterial just like the commercial 45S5 Bioglass® does [16]. Using this glass as a model system, we set to develop new method for nitrate ion removal. In this study, we propose to remove the residual nitrate ions in 70Si30Ca powders stabilized under low temperatures (i.e. 200 ℃ and 350 ℃) by soaking. Deionized water and simulated body fluids (SBF) were selected as soaking medium separately to examine the validity of proposed method.

2. Experimental 2.1. Preparation of 70Si30Ca powders

Tetraethyl orthosilicate (TEOS, purity ≥99.0%), calcium nitrate tetrahydrate (Ca(NO)3-4H2O, purity ≥99.0%) and concentrated nitric acid were purchased from Sigma-Aldrich (Shanghai, China) and used as received.

70Si30Ca bioactive calcium silicate was prepared by sol-gel route. Typical recipe is like the following: 10 mL TEOS was hydrolyzed for 30 min in the mixture of ethanol and water at room temperature with 2 mol/L HNO3 as catalyst. 4.53 g calcium nitrate tetrahydrate was added gradually under continuous stirring. The molar ratio of water to TEOS (R ratio) was 12:1. The obtained clear sol solution was sealed in a polypropylene container and left to gel. The gel was aged at 60 ℃ for a week, followed by drying at 120 ℃ for another week and then stabilized at 200 ℃, 350 ℃ and/or 600 ℃ for 2 h, which were referred as BG-200, BG-350, BG-600, respectively. The stabilized 70Si30Ca calcium silicates were then grounded into powders (<50 mm) for testing.

2.2. Thermal behavior and stabilization simulation

Differential thermal analysis (DTA) and thermal gravimetric analysis (TGA) were employed to study the thermal behavior of 70Si30Ca on a TA Q-600 instrument. The dried gels (obtained before the stabilization stage) were placed in alumina crucibles and measured under a nitrogen flow of 100 mL min-1 using a heating rate of 10 ℃ min-1.

Simulation of the stabilization process was carried out on a Pyris 1 instrument to monitor the mass changes in this stage. The dried gels were placed in platinum crucibles and measured under an air flow of 20 mL min-1 with a heating rate of 10 ℃ min-1. The temperature was kept at 200 ℃, 350 ℃ and/or 600 ℃ for 2 h separately.

2.3. Immersion experiments

150 mg of accurately weighted 70Si30Ca powders were immersed in 100 mL water or simulation body fluid (SBF) at 36.5 ± 0.5 ℃ for 0.5 h, 2 h, 24 h, and 72 h, respectively, to remove the residual nitrate ions.

The nitrate ion remaining in the calcium silicates was determined by FTIR and Raman spectroscopy. FTIR was carried out with a Bruker Equinox 55 instrument in a wave number range from 400 cm-1 to 4000 cm-1 operating in the absorbance mode. Raman was carried out with a Renishaw in Via plus instrument in a laser with the wavelength of 663 nm.

2.4. Evaluation of bioactivity

150 mg of 70Si30Ca samples obtained after above immersion were immersed again in 100 mL SBF at 36.5 ± 0.5 ℃ for 24 h, 72 h and 168 h to examine their in vitro bioactivity. XRD of the powder was employed to examine the formation of hydroxycarbonate apatite (HCA). The solids were collected with filter papers followed by washing with pure water and then dried in vacuum overnight at 40 ℃ before the XRD measurements. The XRD experiments were performed with a Bruker D8 Advance Diffractometer using Cu-Kα radiation (λ = 1.54 Å) and operated at 40 kV and 200 mA, with a step size of 48 min-1 , a counting rate of 30 s per step, and 2θ values from 5° to 80°.

3. Results and discussion

Fig. 1a presents DTA and TGA traces of 70Si30Ca dried gels. DTA trace indicates the heat changes of the sample as a function of temperature in the range of 25-800 ℃, and TGA trace reflects the weight changes. The DTA trace exhibited two obvious endothermic peaks. The first endothermic peak, which initiated at = ℃, corresponds to the loss of physically absorbed water and liquid by-products resulted from the polycondensation reaction. All the water and liquid by-products were removed before 100 ℃ (8.8% weight loss). The second endothermic peak, which initiated at 496 ℃, was caused by the condensation of silanol groups and the decomposition of nitrate groups [12]. The removal of nitrate ions at these temperatures was the main reason of the major weight loss in the TGA trace (31% weight loss). All nitrates were removed by 600 ℃, after which the weight loss was negligible. The total weight loss was 46%. No exothermic peak was observed from 25 ℃ to 800 ℃, indicating no crystallization occurred.

Fig. 1.(a) TGA and DTA traces of 70Si30Ca dried gels performed under N2 protection; (b) simulation of the stabilization process of 70Si30Ca dried gels performed in air atmosphere.

Simulation of the stabilization process of 70Si30Cadried gelswas performed in air atmosphere at a heating rate of 10 ℃min-1 to depict the weight changes in the stabilization stage (Fig. 1b). Similar to the TGA trace, the weight loss before 100 ℃ (about 7.1%) was attributed to the evaporation of physically absorbed water and liquid by-products. And the weight loss occurred after 100 ℃ could be explained by the further condensation of silanol groups and decomposition of nitrate groups. According to the TGA trace and the simulation of stabilization, samples that stabilized at 200 ℃ and 350 ℃ still contained a large amount of nitrate ions to be decomposed. Therefore, 200 ℃ and 350 ℃ were selected to be the stabilization temperatures for the immersion experiments and 600 ℃as the control to evaluate the efficiency of nitrate ion removal.

FTIR and Raman spectra of 70Si30Ca samples before the immersion experiments are shown in Fig. 2. For the FTIR spectra of all glass powders (Fig. 2a), the bands located in the range of 1000- 1250 cm-1 correspond to Si-O-Si asymmetric stretching vibration and the bands at 787 cm-1 are attributed to the Si-O-Si symmetric stretching vibration [17]. The bands in the range of 450-480 cm-1 are ascribed to the Si-O-Si bending mode [18]. Specifically, for BG- 200, the band appeared at 970 cm-1 corresponds to Si-OH bonds [19]. The bands at 740 cm-1, 825 cm-1, 1359 cm-1, 1380 cm-1, and 1438 cm-1 are characteristic of calcium nitrate. For the Raman spectra of all glass powders (Fig. 2b), only one peak at 1052 cm-1 representing the nitrate ion is observed. FTIR and Raman both reflect the relative content of nitrate groups in samples. It is obvious that BG-350 contained less calcium nitrate than BG-200 while no nitrate ions were detected for BG-600 (Fig. 2a and b). And almost all nitrate ions could be removed from the solids after soaked for 0.5 h both in deionized water and SBF (Fig. 3a and b).

Fig. 2.The spectra of original 70Si30Ca stabilized at different temperatures: (a) FTIR; (b) Raman.

Fig. 3.The FTIR spectra of original 70Si30Ca stabilized at different temperatures after immersed in different solution for different time period: (a) in water; (b) in SBF.

SBF is a solution that has almost identical ion concentrations with the human blood plasma (HBP), and the hydroxyapatite formation on the surface of the materials after immersed in SBF have been used as a criterion for the test of the bioactivity in vitro [20]. The immersed solids were immersed again in SBF for another 24 h, 72 h and 168 h to examine their in vitro bioactivity left after the removal of nitrate ions. All the calcium silicates still are amorphous and have no sharp peak appeared after being immersed in SBF and water for 0.5 h (Figs. 4 and 5). For that after immersion in SBF, the XRD confirmed the appearance of sharp peaks when the solids were being immersed again in SBF for 24 h and 72 h indicating the formation of hydroxyapatite (Fig. 4a and b), and which is similar to the samples treated at 600 ℃ (Fig. 4c), thereby proving their in vitro bioactivity. For that after immersion in water, no hydroxyapatite was detected when the solids were immersed again in SBF for 24 h and 72 h (data not shown), and few hydroxyapatite formed on the surface of the solids when being immersed for 168 h (Fig. 5a and b), indicating a decrease in bioactivity. This may because Ca2+, including those loosely bonded to the silica network and those combined with nitrate ions, was also released from the samples. And also, it could be found that the samples stabilized at higher temperature have much more hydroxyapatite formation at the same condition (Figs. 4 and 5), probably because much more calcium is incorporated into the silica network at higher temperature (350 ℃) [21].

Fig. 4.XRD of BG-200 and BG-350 (that have been immersed in SBF for 0.5 h) after being immersed in SBF again for different time comparing with the samples treated at 600 8C (BG-600).

Fig. 5.XRD of BG-200 and BG-350 (that have been immersed in water for 0.5 h) after being immersed in SBF again for 168 h.
4. Conclusion

An immersion route for the rapid removal of residual nitrate ions in powders of low-temperature stabilized calcium silicates was established with soaking medium of deionized water and SBF. It was demonstrated that nitrate ions could be removed in 0.5 h from the powders by immersion, and the solids were still bioactive especially after SBF medium immersion. This work provides an alternative way of nitrate ion removal instead of thermal decomposition, thus can lower the sample processing temperature to a degree that most polymer moieties can survive.


This work was supported by NSFC (Nos. 81470101, 51173193) and Royal Society/Natural Science Foundation of China international exchange (No. 51411130151).

[1] P. Sepulveda, J.R. Jones, L.L. Hench, Bioactive sol-gel foams for tissue repair, J. Biomed. Mater. Res. 59(2002) 340-348.
[2] L.L. Hench, Bioceramics:from concept to clinic, J. Am. Ceram. Soc. 74(1991) 1487-1510.
[3] L.L. Hench, The story of Bioglass®, J. Mater. Sci. Mater. Med. 17(2006) 967-968.
[4] L.L. Hench, R.J. Splinter, W.C. Allen, T.K. Greenle, Bonding mechanisms at the interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 5(1971) 117-141.
[5] P. Saravanapavan, L.L. Hench, Mesoporous calcium silicate glasses. I. Synthesis, J. Non-Cryst. Solids 318(2003) 1-13.
[6] A.L. Li, D. Qiu, Phytic acid derived bioactive CaO-P2O5-SiO2 gel-glasses, J. Mater. Sci. Mater. Med. 22(2011) 2685-2691.
[7] E.M. Valliant, C.A. Turdean-Ionescu, J.V. Hanna, M.E. Smith, J.R. Jones, Role of pH and temperature on silica network formation and calcium incorporation into sol-gel derived bioactive glasses, J. Mater. Chem. 22(2012) 1613-1619.
[8] M.M. Pereira, J.R. Jones, R.L. Orefice, L.L. Hench, Preparation of bioactive glasspolyvinyl alcohol hybrid foams by the sol-gel method, J. Mater. Sci. Mater. Med. 16(2005) 1045-1050.
[9] L.C. Bandeira, K.J. Ciuffi, P.S. Calefi, E.J. Nassar, Silica matrix doped with calcium and phosphate by sol-gel, Adv. Biosci. Biotechnol. 1(2010) 200-207.
[10] A. Mori, C. Ohtsuki, T. Miyazaki, et al., Synthesis of bioactive PMMA bone cement via modification with methacryloxypropyltri-methoxysilane and calcium acetate, J. Mater. Sci. Mater. Med. 16(2005) 713-718.
[11] S. Lin, C. Ionescu, K.J. Pike, M.E. Smith, J.R. Jones, Nanostructure evolution and calcium distribution in sol-gel derived bioactive glass, J. Mater. Chem. 19(2009) 1276-1282.
[12] J.R. Jones, L.M. Ehrenfried, L.L. Hench, Optimising bioactive glass scaffolds for bone tissue engineering, Biomaterials 27(2006) 964-973.
[13] M.Y. Koh, H.M. Kim, C. Ohtsuki, Synthesis of a bi-structured hybrid in a CaO-SiO2-PTMO system and in vitro evaluation on its potential of bone-bonding property, Mater. Sci. Eng. 30(2010) 454-459.
[14] Y.S. Sun, A.L. Li, F.J. Xu, D. Qiu, A low-temperature sol-gel route for the synthesis of bioactive calcium silicates, Chin. Chem. Lett. 24(2013) 170-172.
[15] A.L. Li, H. Shen, H.H. Ren, et al., Bioactive organic/inorganic hybrids with improved mechanical performance, J. Mater. Chem. B 3(2015) 1390-1397.
[16] P. Saravanapavan, J.R. Jones, S. Verrier, et al., Binary CaO-SiO2 gel-glasses for biomedical applications, Biomed. Mater. Eng. 14(2004) 467-486.
[17] S. Falaize, S. Radin, P. Ducheyne, In vitro behavior of silica-based xerogels intended as controlled release carriers, J. Am. Ceram. Soc. 82(1999) 969-976.
[18] A.M. El-Kady, A.F. Ali, R.A. Rizk, M.M. Ahmed, Synthesis, characterization and microbiological response of silver doped bioactive glass nanoparticles, Ceram. Int. 38(2012) 177-188.
[19] J.P. Zhong, D.C. Greenspan, Processing and properties of sol-gel bioactive glasses, J. Biomed. Mater. Res. 53(2000) 694-701.
[20] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W3, J. Biomed. Mater. Res. 24(1990) 721-734.
[21] R.J. Newport, L.J. Skipper, D. Carta, et al., The use of advanced diffraction methods in the study of the structure of a bioactive calcia:silica sol-gel glass, J. Mater. Sci. Mater. Med. 17(2006) 1003-1010.