In vitro bioactivity of biphasic calcium phosphate silicate glass-ceramic in CaO-SiO 2P 2 O 5 system

The main purpose of the paper is the evaluation of the influence of chemical composition of the gel of the synthesized 15CaO·0.5P2O5·6SiO2 glass-ceramic on the structure, crystallization behaviour and in vitro bioactivity in static conditions for different periods of time 3, 9 and 30 days in 1.5SBF. The obtained glass-ceramic was synthesized via polystep sol-gel technique. The structure of the prepared and the one thermally treated at 1200°C for 2 h powder was studied by XRD, 29Si MAS NMR, FTIR and SEM.

Analyses of the chemical and phase composition of glass-ceramic materials in the CaO-SiO 2 -P 2 O 5 system, obtained by different research groups can be summarized in Table 1.
The effect of compositional changes on the in vitro [7][8][9]20,22,23], in vivo [6,10,[17][18][19] bioactivity and other properties [11][12][13][14][15][16] have also been investigated.Margussian et al. [7] found that after one month immersion in SBF the hydroxyapatite (HA) layer can be formed on the glass-ceramic surface.They also concluded that the decrease of P 2 O 5 , in coincidence with [9,20], enhanced the bioactivity.Salinas et al. [8] studied that after chemical treatment of the prepared glassceramics with 1 M HCl, the in vitro studies showed the formation of an apatite-like layer covering certain areas of the material surface.In this context Pietl et al. [9] postulated that the CO 3 HA can be formed on the glass-ceramic surface.On the base of these results, they concluded that the CO 3 HA layer formation of these compositions in in vitro tests is much faster than in commercial bioactive materials such as synthetic hydroxyapatite ceramic, A/W glass-ceramic, Ceravital and Bioverit, for which the onset time usually takes at least seven days.Radev et al. [22] observed that the synthesized ceramic material with chemical composition 15CaO•P 2 O 5 •6SiO 2 demonstrates in vitro bioactivity in 1.5 SBF.The observed CO 3 HA layer had a "flower-like" morphology.Radev et al. [23] also investigated that CaO-SiO 2 -P 2 O 5 glass-ceramics doped with MgO has shown in vitro bioactivity.They observed two types of morphologies of CO 3 HA layer on the prepared samples -longitudinal dendrite arms and irregular assemblies of particles.In this paper, we investigated the influence of composition of the prepared glass-ceramic with chemical composition 15CaO•0.5P 2 O 5 •6SiO 2 on the structure, crystallization behaviour and in vitro bioactivity in static conditions for different periods of time.

The synthesis of the ceramic sample
The ceramic material in the CaO-SiO 2 -P 2 O 5 system has been synthesized via polystep sol-gel method.The chemical composition of the obtained sample is described as 66.1 CaO, 28.3 SiO 2 and 5.6 P 2 O 5 (wt.%).
The procedure for the synthesis of the ceramic material with chemical composition of the gel 15CaO•0.5P 2 O 5 •6SiO 2 is similar to this detail documented in ref. 22.The obtained mixed (calcium phosphate-SiO 2 ) sol was gelated at 120°C for 12 hours and thermally treated at 1200°C for 2 hours in tubular furnace.

In vitro test for bioactivity in static conditions
Bioactivity of obtained ceramic materials was evaluated by examining the apatite formation on their surfaces in 1.5SBF (simulated body fluid).The 1.5SBF solution was prepared from reagents as follows: NaCl = 11.9925g, NaHCO 3 = 0.5295 g, KCl = 0.3360 g, K 2 HPO 4 •3H 2 O = 0.3420 g, MgCl 2 •6H 2 O = 0.4575 g, CaCl 2 •2H 2 O = 0.5520 g, Na 2 SO 4 = 0.1065 g, and buffering at pH 7.4 at 36.5°C with TRIS=9.0075g and 1M HCl in distilled water.The synthesized ceramic powder was pressed at 50 MPa with PVA to obtain disc specimens (12 mm diameter and 2 mm thick) and immersed in 1.5SBF at the human body temperature (36.6°C) in polyethylene bottles in static conditions for 3, 9 and 30 days.A few drops of 0.5% sodium azide (NaN 3 ) was added to the 1.5 SBF solution to inhibit the growth of bacteria [24,25].After soaking, the specimens were removed from the fluid and gently rinsed with distillated water, and then dried at 36.6°C for 12 h.

Methods for analysis
The structure and in vitro bioactivity of ceramic materials was monitored by X-ray diffraction (XRD) analysis, Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy with X-ray microanalysis (SEM/EDS).
Powder X-ray diffraction spectra were collected within the range from 10° to 80° 2θ with a constant step 0.04° 2θ and counting time 1 s/step on a Bruker D8 Advance diffractometer with CuK α radiation and SolX detector.The spectra were evaluated with the Diffracplus EVA package.
FTIR transmission spectra for the obtained hybrids were recorded by using a Bruker Tensor 27 spectrometer with scanner velocity 10 kHz.KBr pellets were prepared by mixing ~1 mg of the samples with 300 mg KBr.Transmission spectra were recorded using MCT detector, with 64 scans and 1 cm -1 resolution.SEM (Jeol, JSM-35 CF, Japan) in conijunction with EDS was used to ascertain the morphology and chemical constituents of the prepared hybrids after immersion in 1.5 SBF for 3 days at accelerating voltage of 15 kV.

29
Si magic-angle spinning (MAS) NMR spectra were recorded at 79.49 MHz on a 9.4 T Bruker Avance 400 spectrometer, using 408 pulses, a recycle delay of 60 s and a spinning rate of 5 kHz.

29
Si MAS NMR spectroscopy in the solid state provides significant information about the different silicon species that constitute the structure formed, as well as the degree of condensation between the silicate sheets in the structure of the hybrid.The Q n notation is convenient for indicating the number of n of -O-Si link- Fig. 3 shows FTIR spectrum for the obtained sample after thermal treatment at 1200°C for 2 hours.In the presented spectrum the absorption bands of silicate groups were clearly evident.The intense band centred at 990 cm -1 was assigned to the Si-O-Si asymmetric stretch [29], the bands at 940 and 925 cm -1 to the Si-O symmetric stretch [29][30][31][32] and the bands at 551, 521 and 460 cm -1 to the Si-O-Si vibrational mode of bending [29,30,33,34].The band, centred at 521, 551, 630 and 1011 cm -1 can be assigned to the presence of ν 3 SiO 4 or ν 4 PO 4 groups [30,31,[35][36][37][38]. Around 720 cm -1 in the prepared sample is the Fig. 4 presents the FTIR data after immersion of the prepared ceramic in 1.5 SBF for different periods of time -3, 9 and 30 days.As it can be seen, Fig. 4 shows the infrared absorption spectra of carbonate containing hydroxyapatite (CO 3 HA) formed on the surface of the immersed samples for 3, 9 and 30 days.The 1 mode of PO 4 3-ion is represented in apatites by a very narrow band (944 cm -1 , curves a, 6 Fig. 3 FTIR spectrum for calcium phosphate silicate obtained after heating at 1200 o C for 2 hours In the presented spectrum the absorption bands of silicate groups were clearly evident.The intense band centered at 990 cm -1 was assigned to the Si-O-Si asymmetric stretch [29], the bands at 940 and 925 cm -1 to the Si-O symmetric stretch [29][30][31][32] and the bands at 551, 521 and 460 cm -1 to the Si-O-Si vibrational mode of bending [29,30,33,34].The band, centered at 521, 551, 630 and 1011 cm -1 can be assigned to the presence of 3 SiO 4 or 4 PO 4 groups [30,31,[35][36][37][38]. Around 720 cm -1 in the prepared sample is the band due to Si-O bending vibrations [39].The important vibrations of the PO 4 3-can be distinguished in the three main regions: (i) a sharp peak at 1058 cm -1 associated with asymmetric stretching vibration [40]; (ii) a weak bands at 940 and 925 cm -1 corresponding to the symmetric stretching [38,40,41]; (iii) two well resolved peaks at 551 and 525 cm -1 assigned to the asymmetric bending modes [42][43][44].In our synthesized sample, however, the characteristic bands with small intensity, posited at 1011 and 940 cm -1 could be ascribed to the presence of -tricalcium phosphate ( -TCP) in accordance with Ooi et al. [45] and de Aza et al. [46,47] .Furthermore, in keeping with Z. Gou et al. [29] the absorption bands, posited at 997, 940 and 521 cm -1 , respectively could be ascribed to the presence with Ca 2 SiO 4 .The double peaks at 2364 and 2334 cm -1 assigned to the O-H stretch vibration of hydrogen bonded O-H groups, together with the peak at 1638 cm -1 ascribed to the O-H bending mode are evidences of the presence of adsorbed water in the prepared ceramic powder [48].Surprisingly, as it can be seen from the presented Fig. 4, we can observe the bands, centered at 1745, 1460, 1428, 1376 and 1320 cm -1 .The assignments for CO 3 2-in the prepared ceramic powder are 3 (asymmetric stretching) at 1460, 1428 and 1376 cm -1 and 2 (bending) at 1320 cm -1 [36,[49][50][51].The band centered at 1750 cm - 1 can be attributed to the presence of small amount of calcite [52].The presence of carbonate bands are attributed to a carbonation process of the material due to the atmospheric CO 2 as a consequence of the high calcium content in the prepared sample.band due to Si-O bending vibrations [39].The important vibrations of the PO 4 3-can be distinguished in the three main regions: i) a sharp peak at 1058 cm -1 associated with asymmetric stretching vibration [40]; ii) a weak bands at 940 and 925 cm -1 corresponding to the symmetric stretching [38,40,41]; iii) two well resolved peaks at 551 and 525 cm -1 assigned to the asymmetric bending modes [42][43][44].In our synthesized sample, however, the characteristic bands with small intensity, posited at 1011 and 940 cm -1 could be ascribed to the presence of β-tricalcium phosphate (β-TCP) in accordance with Ooi et al. [45] and de Aza et al. [46,47].Furthermore, in keeping with Gou et al. [29] the absorption bands, posited at 997, 940 and 521 cm -1 , respectively could be ascribed to the presence of Ca 2 SiO 4 .The double peaks at 2364 and 2334 cm -1 assigned to the O-H stretch vibration of hydrogen bonded O-H groups, together with the peak at 1638 cm -1 ascribed to the O-H bending mode are evidences of the presence of adsorbed water in the prepared ceramic powder [48].Surprisingly, as it can be seen from the presented Fig. 3, we can observe the bands, centred at 1745, 1460, 1428, 1376 and 1320 cm -1 .The assignments for CO 3 2-in the prepared ceramic powder are ν 3 (asymmetric stretching) at 1460, 1428 and 1376 cm -1 and ν 2 (bending) at 1320 cm -1 [36,[49][50][51].
The band centred at 1750 cm -1 can be attributed to the presence of small amount of calcite [52].The presence of carbonate bands is attributed to a carbonation process of the material due to the atmospheric CO 2 as a consequence of the high calcium content in the prepared sample.The obtained results are in a good correspondence with those, published by Martinez et al. [53] for CaO-SiO 2 sol-gel glasses.Moreover, the spectrum do not display a number of very weak absorption bands at 2900-2800, 1644 and 878 cm −1 .According to Zaki et al. [54,55] these bands are indicative for pure CaO.
Fig. 4 presents the FTIR data after immersion of the prepared ceramic in 1.5SBF for different periods of time -3, 9 and 30 days As it can be seen, Fig. 4 shows the infrared absorption spectra of carbonate containing hydroxyapatite (CO 3 HA) formed on the surface of the immersed samples for 3, 9 and 30 days.The ν 1 mode of PO 4 3-ion is represented in apatite by a very narrow band at 944 cm -1 and the ν 2 mode produces an absorption peak, centred at 476 cm -1 (Fig. 4, curves a and c) and 461 cm -1 (Fig. 4, curve b) [56][57][58][59][60].As shown by other authors, the main absorbance signal of PO 4 3-appears in the triply degenerate ν 3 domain [58].Moreover, the ν 4 mode gives a well defined peak, posited at 564 cm -1 [58,60].The broad peak at around 1000 cm -1 (1050 cm -1 for all three samples) is due to both the ν 1ν 3 PO 4 3-modes and the most intense SiO 4 4-absorption band [58][59][60].The ν 2 signal is also masked by another Si-O-Si peak, and the only well defined PO 4 3-band is thus the doubly degenerate ν 4 mode.Among the four internal vibrational modes of the free CO 3 2-ion, only two are of importance for FTIR investigations in calcium phosphates: ν 2 and ν 3 [61].In the presented FTIR spectra, the absence of the C-O absorption bands at ~710 cm -1 indicates that no detectable calcite or aragonite is associated with our CO 3 HA over the synthesized sample after static in vitro test.This result confirms our X-ray diffraction analysis, depicted on Fig. 6.On the one hand, Feki et al. [62] postulated that the absorption band, posited at 720 cm -1 could be assigned to the presence of B-substitution (CO 3 2-→PO 4 3-) into hydroxyapatite lattice.On the other hand, the weak bands centred at ~876, ~1480 and ~1423 cm -1 could be ascribed to the B-substitution into hydroxyapatite lattice, as already mentioned in literature [59][60][61]63].
The more detailed information about the type of substitution in CO 3 HA, formed on the surface of the prepared ceramic, after 30 days immersion in 1.5SBF, was analyzed by curve fitting in the region 1850-1250 cm -1 (Fig. 5).It can be seen that the bands centred at 1481, 1427 and 1400 cm -1 could be ascribed to the presence of B-type carbonate substitution, i. e. part of PO 4  2-[53,61,64-68].As indicated earlier by Fleet et al. [69] the peak, posited at 1514 cm -1 can be related to the presence of A-type substitution (CO 3 2-→OH -) into hydroxyapatite lattice.XRD of the synthesized sample after 3 days immersion in 1.5SBF is given in Fig. 6.From depicted XRD patterns it can be seen that the obtained samples do not undergo drastic changes after 3 days immersion in 1.5SBF.As can be seen, the mean diffrac-tion peaks of CO 3 HA (PDF 04-0697) are presented.The obtained XRD results are in accordance with presented FTIR data.
SEM of the prepared calcium phosphate silicate ceramic powder, after thermal treatment at 1200°C for 2 hours, clearly depicts (Fig. 7b) that the synthesized sample has a plate-like morphology.This morphology is characteristic for the silicon substituted apatites as denoted in [70].From the presented SEM images it can also be seen that the prepared powder has none fully densified structure.Some authors postulate that the preparation of this structure could be assigned to the presence of silica in calcium phosphate structure and the temperature of thermal treatment of dried calcium phosphate silicate gel [71].
SEM images for the samples after in vitro test for 9 and 30 days are given in Figs. 8 and 9.After immersion of the prepared ceramics for different periods of time, it is observed (Figs.8a and 9a) that the surface of the immersed samples is fully covered with the particles with different morphology.When the ceramic was immersed for 9 days in 1.5-SBF in static conditions, SEM showed that on the surface are deposited some spherical particles (Fig. 8b,c).The diameters of these spheres were 1-3 µm.At low magnification (Fig. 8b), it may seem to us that the spheres had crackles on their bumpy surface.But it is not so.At high magnification (Fig. 8d) it can be seen that the big spherical particles composed of small nanospheres.The diameter of these spheres riches up to 0.2 µm.After 30 day immersion in 1.5SBF in static conditions, SEM depicts that a dense deposits cover the entire surface of the prepared ceramic.As can be seen from Fig. 7b such deposits are not observed prior in vitro test in static conditions.At high magnification (Fig. 9c,d) we can also see that the spherical particles are accumulated on the ceramic surface.As it has been explained above, these spheres are consisted of the little spheres.These spheres have a diameter of 0.1-0.2µm

IV. Conclusions
A new biphasic calcium phosphate silicate ceramic material has been synthesized via polystep solgel method.After thermal treatment of the dried gel at 1200 o C for 2 h, XRD showed the presence of two phases -Ca 15 (PO 4 ) 2 (SiO 4 ) 6 and Ca 2 SiO 4 •0.05Ca 3 (PO 4 ) 2 FTIR study confirmed XRD data. 29SiMAS NMR indicated that the obtained material has only Q o (-72.1 ppm) silicon environments (=Si(O-) 4 ).In vitro evaluation of bioactivity was carried out in static conditions in 1.5SBF.FTIR spectra showed that the increase in the intensity of CO 3 2-is associated with carbonate containing apatite (CO 3 HA) formation on the surface.SEM images demonstrate that the apatite phase covered the entire surface after 30 days immersion in

Figure 2 .
Figure 2. 29 Si MAS NMR for the thermally treated at 1200°C for 2 hours glass-ceramic

Fig. 4
Fig.4FTIR spectra of the samples after in vitro test for bioactivity in static conditions for 3days (a), 9 days (b) and 30 days (c)As it can be seen, Fig.4shows the infrared absorption spectra of carbonate containing hydroxyapatite (CO 3 HA) formed on the surface of the immersed samples for 3, 9 and 30 days.The 1 mode of PO 4 3-ion is represented in apatites by a very narrow band (944 cm -1 , curves a, Fig. 3 FTIR spectrum for calcium phosphate silicate obtained after heating at 1200 o C for 2 hoursIn the presented spectrum the absorption bands of silicate groups were clearly evident.The intense band centered at 990 cm -1 was assigned to the Si-O-Si asymmetric stretch[29], the bands at 940 and 925 cm -1 to the Si-O symmetric stretch[29][30][31][32] and the bands at 551, 521 and 460 cm -1 to the Si-O-Si vibrational mode of bending[29,30, 33, 34].The band, centered at 521, 551, 630 and 1011 cm -1 can be assigned to the presence of 3 SiO 4 or 4 PO 4 groups[30, 31,[35][36][37][38].Around 720 cm -1 in the prepared sample is the band due to Si-O bending vibrations[39].The important vibrations of the PO 4 3-can be distinguished in the three main regions: (i) a sharp peak at 1058 cm -1 associated with asymmetric stretching vibration[40]; (ii) a weak bands at 940 and 925 cm -1 corresponding to the symmetric stretching[38,40, 41]; (iii) two well resolved peaks at 551 and 525 cm -1 assigned to the asymmetric bending modes[42][43][44].In our synthesized sample, however, the characteristic bands with small intensity, posited at 1011 and 940 cm -1 could be ascribed to the presence of -tricalcium phosphate ( -TCP) in accordance withOoi et al. [45]  and de Aza et al.[46, 47]  .Furthermore, in keeping with Z.Gou et al. [29]  the absorption bands, posited at 997, 940 and 521 cm -1 , respectively could be ascribed to the presence with Ca 2 SiO 4 .The double peaks at 2364 and 2334 cm -1 assigned to the O-H stretch vibration of hydrogen bonded O-H groups, together with the peak at 1638 cm -1 ascribed to the O-H bending mode are evidences of the presence of adsorbed water in the prepared ceramic powder[48].Surprisingly, as it can be seen from the presented Fig.4, we can observe the bands, centered at 1745, 1460, 1428, 1376 and 1320 cm -1 .The assignments for CO 3 2-in the prepared ceramic powder are 3 (asymmetric stretching) at 1460, 1428 and 1376 cm -1 and 2 (bending) at 1320 cm -1[36,[49][50][51].The band centered at 1750 cm - 1 can be attributed to the presence of small amount of calcite[52].The presence of carbonate bands are attributed to a carbonation process of the material due to the atmospheric CO 2 as a consequence of the high calcium content in the prepared sample.The obtained results are in a good correspondence with those, published from Martinez et al. for CaO-SiO 2 sol-gel glasses[53].Moreover, the spectrum do not display a number of very weak absorption bands at 2900-2800, 1644 and 878 cm 1 .According to Zaki et al. these bands are indicative for pureCaO [54, 55].

Figure 3 .
Figure 3. FTIR spectrum for calcium phosphate silicate obtained after heating at 1200°C for 2 hours

Figure 6 .
Figure 5 Curve fitting spectra of the type of CO 3 HA, formed on the ceramic sample after 30 days immersion in 1.5SBF

Figure 7 .Figure 8 .
Figure 7. SEM of the synthesized and thermally treated ceramic powder at different magnifications before in vitro test

Figure 9 .
Figure 9. SEM of the synthesized sample after 30 days immersion in 1.5SBF at different magnifications