Bioactive ceramic powder prepared using a new sol-gel process

Bioactive ceramic powder containing 45% SiO2, 24.5% CaO, 24.5% Na2O and 6% P2O5 (wt.%) was synthesized using a new sol-gel process. The prepared material was investigated by several physico-chemical methods. The X-ray diffraction showed the presence of two crystalline phases, Na6Ca3Si6O18 and NaCaPO4. The high specific surface area (47 m/g) and a mesoporous structure with the average pore size of 2.6 nm were confirmed using low-temperature nitrogen adsorption technique. In vitro experiments were carried out by soaking the powder sample in simulated body fluid (SBF) at different times. XRD, FTIR and SEM coupled with EDS were used for in vitro evaluation of bioactivity. The obtained results confirmed that the ceramic powder can be used as biomaterial for bone tissue engineering due to its high bioactivity expressed by the rapid formation of a biological carbonated hydroxyapatite layer on its surface after in vitro assay.


I. Introduction
Bioactive glasses are traditionally processed by melting method, in which precursor materials are melted at high temperature.The obtained liquid is quenched to form an amorphous material at room temperature.The first melt-derived bioglass 45S5 containing 45% SiO 2 , 24.5% CaO, 24.5% Na 2 O and 6% P 2 O 5 (wt.%) was prepared by Hench in 1969 [1][2][3][4].Recently, the solgel method has been widely used to prepare bioglasses, due to its higher surface area in comparison to the meltderived bioglass [5][6][7].The high surface area of the solgel bioglass enhances the interfacial reactions between the material and physiological environment that accelerates the formation of biological apatite layer.Many bioglasses have been formed by sol-gel method, but a similar glass to the 45S5 one has not been synthesized successfully yet because of its crystallization during sol-gel procedure [8].Several studies reported the preparation of quaternary SiO 2 -CaO-Na 2 O-P 2 O 5 bioglasses by solgel method, but their compositions were very different from the 45S5 bioglass [9][10][11].While the 45S5 bioglass prepared by sol-gel process has not been observed yet, an alternative approach has to be considered for synthe-sis of a similar quaternary system with high bioactivity.Thus, in this study we reported a new sol-gel process to prepare a ceramic powder having the same composition as the bioglass 45S5.This ceramic powder revealed the high bioactivity after in vitro assay in SBF solution.

Powder preparation
The bioactive ceramic powder was prepared by mixing 6.242 g of TEOS and 0.616 g of TEP in 15 ml of distilled water and stirring for 2 hours by magnetic agitation at 500 rpm.The pH value of the mixture was adjusted to 1.5 using HNO 3 solution.After that 4.127 g of Ca(NO 3 ) 2 ×4 H 2 O and 2.688 g of NaNO 3 were respectively added to the mixture at 60-minute intervals.Fi-nally, 3 drops of 30% ammonia solution were poured into the mixture and continuously stirred for 4 hours to obtain a condensed gel.The resulting gel was frozen and then freeze-dried at −60 °C for 24 hours to remove the residual water and ethanol.The obtained dry gel was heated at 1000 °C for 2 hours and converted into ceramic powder.The synthesis protocols are summarized in Fig. 1.

In vitro bioactive test
In vitro bioactivity of the ceramic powder was investigated by soaking 100 mg of material in 200 ml of SBF solution at body temperature.The SBF composition is similar to that of human blood plasma.It was prepared by dissolving NaCl, NaHCO 3 , KCl, K 2 HPO 3 ×3 H 2 O, MgCl 2 ×6 H 2 O and CaCl 2 in distilled water and buffered with (CH 2 OH) 3 CNH 2 and HCl (6 N) to adjust the pH value at 7.4 according to the Kokubo's method [12].

Physico-chemical charaterizations
The ceramic powders before and after soaking in SBF solution were investigated by using several physicochemical techniques.The specific surface area and porosity of ceramic powder were determined by the low-temperature nitrogen adsorption-desorption measurements on multi-point BET, ASAP 2010 Analyzer.In order to identify the crystalline phases of synthetic material and evaluate the formation of apatite layer after in vitro assay, X-ray diffraction (XRD) measurements were realized on Bruker D8 Advance diffractometer.Powder samples were mixed homogeneously with cyclohexane and dropped on the surfaces of plastic tablets.Then, these tablets were dried to remove the solvent and introduced into diffractometer.The XRD data were acquired in the range of 10-70°(2θ) with a scanning speed of 1°/min.The Fourier transformed infrared spectroscopy (FTIR) (Bruker Equinox 55) was employed to identify the functional groups of the ceramic powder before and after in vitro assay.Powder materials were ground and mixed thoroughly with KBr powder in the ratio 1:100.FTIR spectra were recorded in the wavenumber range of 400 and 4000 cm -1 , at a resolution of 2 cm -1 .Scanning electron microscopy (SEM) (JEOL JSM 6301) coupled with energy dispersive X-ray spectroscopy (EDS) was used to evaluate the morphological surface of ceramic powder as a function of soaking time in SBF solution and determine the Ca/P elemental ratio in the apatite layer after in vitro assay.

Porosity characterization
The nitrogen adsorption and desorption isotherm curve of the bioactive powder is presented in Fig. 2. It exhibited a type IV character according to the IUPAC classification, typical for a mesoporous material containing the pores between 2 and 50 nm in diameter.The specific surface area, pore volume and pore size of the ceramic powder are listed in Table 1.It can be seen that the specific surface area of the sol-gel derived ceramic powder is much higher than that obtained for the meltderived glass 45S5 [6].

Phase composition
Figure 3 shows the XRD diagrams of the ceramic powder before and after soaking in SBF solution.The  identification of crystalline phases of synthetic ceramic was performed using the literature data [13][14][15] and the XRD diagram of hydroxyapatite (HA pattern in Fig. 3) was also presented for comparison [16].The XRD pattern of the annealed ceramic powder exhibited two crystalline phases.The major crystalline phase is Na 6 Ca 3 Si 6 O 18 , which is close to Na 2 Ca 2 Si 3 O 9 phase found in the heat treated 45S5 bioactive glass [17,18].The minor phase is NaCaPO 4 and its existence illustrated the presence of phosphorus ions in the ceramic composition adapting the chemical stoichiometry of the initial synthetic system.After 3 days of soaking in SBF solution, the characteristic peaks of Na 6 Ca 3 Si 6 O 18 phase become weaker or disappear while two new peaks at 26°and 32°( 2θ) emerge in the diffractogram of the ceramic powder.These new peaks correspond to the (002) and (211) planes in the structure of hydroxyapatite crystals, respectively.The obtained results confirmed bioactivity of the ceramic powder.The disappearance of Na 6 Ca 3 Si 6 O 18 diffraction peaks continues while the intensities of two apatite characteristic peaks are not much modified with an increase in the soaking time from 3 days to 15 days.This result indicated that the apatite formation on the surface of ceramic powder could be completed almost after only 3 days of immersion.

IR spectroscopy analysis
Figure 4 shows the FTIR spectra of the ceramic powder before and after soaking in SBF solution.The identification of functional bands referred to previous studies [10,17,18].The IR spectrum of synthetic hydroxyapatite (HA spectra in Fig. 4) was used as a reference to identify the formation of apatite phase on the surface of the ceramic particles as function of soaking time [19].The FTIR spectrum of the ceramic powder presents the characteristic bands of a phospho-silicate network.Three bands at 1094, 730 and 690 cm -1 are attributed to the symmetric stretching Si-O-Si vibrations.The band at 1035 cm -1 is assigned to the asymmetric stretching Si-O-Si vibration.The band at 920 cm -1 corresponds to Si- After soaking in SBF solution, the initial structure of the ceramic powder changed significantly due to the interfacial interactions between the material and the physiological solution.Consequently, the spectrum of the ceramic powder contains four new well-defined phosphate bands at 565, 603, 960 and 1039 cm -1 after 3 days of soaking in SBF solution.They are assigned to the stretching vibrations of PO 4 3-groups in apatite crystalline phase.Two carbonate bands at 874 and 1420 cm -1 are also observed.The obtained results confirmed the formation of a carbonated hydroxyapatite layer on the surface of the ceramic particles after in vitro assay.In addition, the IR spectrum of the ceramic powder soaked in SBF exhibits three new bands of Si-O-Si at 470 cm -1 , 799 cm -1 (bending vibration) and 1070 cm -1 (stretching vibration).They are characteristic for the pure silica [4].This result confirmed the formation of a SiO 2 gel on the surface of the ceramic particles and proposed interactive mechanism between bioactive glass and physiological solution described by Hench [1][2][3].It is summarized in following several steps: (1) rapid exchange of protonated H 3 O + from the physiological solution with Ca 2+ , Na + ions in bioglass to form the Si-OH groups, (2) loss of soluble silica as Si(OH) 4 by breaking Si-O-Si bridging links and subsequent formation of silanol groups at the surface in this process, (3) condensation and repolymerization of surface silanols to form SiO 2rich surface layer, (4) migration of Ca 2+ and PO 4 3-from bioglass through the surface of the silica-rich layer and deposition of these ions from SBF solution to form an amorphous calcium phosphate (ACP) film on top of the SiO 2 -rich layer, (5) incorporation of OH -, CO 3 2-from the solution and subsequent crystallization of the ACP layer to form a hydroxycarbonate apatite layer (HCA).
After 15 days of soaking in SBF solution, the intensities of phosphate characteristic bands at 565, 603, 960 and 1039 cm -1 did not change much.This result is fully in accordance with the XRD analyses, in which the characteristic peaks of hydroxyapatite did not change much from 3 to 15 days of immersion.The apatite layer could be formed completely on the surface of the ceramic particles after 3 days of soaking in SBF solution.On the other hand, the characteristic bands of silica gel become more intensive after 15 days of immersion.This confirmed the growth of silica layer as a function of the soaking time in SBF liquid.

SEM and EDS analysis
Figure 5 shows SEM images of the ceramic powder before and after soaking in SBF solution.The surface of the ceramic particles before soaking was smooth.After immersion in SBF solution, it was modified strongly due to the interfacial chemical reactions between the ceramic powder and the physiological solution.After 1 day of immersion, the small particles began to form, but the surface of the ceramic particles was not much mod- ified.After 3 days of immersion, the surface of the ceramic particles was covered completely with a homogeneous layer with apatite crystalline structure (according to the XRD and FTIR results).After 15 days of immersion, the surface of the ceramic particles changes only a little bit more, compared to that obtained after 3 days soaking.The above results confirmed the high bioactivity of the synthetic ceramic by formation of a dense and visible apatite layer after only 3 days in SBF solution.In previous studies [20,21], a series of B 2 O 3 -CaO-SiO 2 -P 2 O 5 showed their bioactivity through the creation of apatite particles after 3 days and a complete apatite layer formation after 7 days of immersion in SBF fluid.
The EDS spectrum of the ceramic powder after 15 days of immersion in SBF solution is shown in Fig. 6.It shows the presence of Na, Si, P, Cl, Ca and O elements.The obtained Ca/P ratio was of 1.61.It was nearly equal to the chemical stoichiometry of hydroxyapatite (Ca/P = 1.67).This result confirmed also the formation of bonelike apatite on the surface of the ceramic particles.

IV. Conclusions
Bioactive ceramic powder containing 45% SiO 2 , 24.5% CaO, 24.5% Na 2 O and 6% P 2 O 5 (wt.%) has been successfully prepared using a new sol-gel process.It consisted of two crystalline phases: Na 6 Ca 3 Si 6 O 18 and NaCaPO 4 and has a high specific surface area.The in vitro assay was carried out by soaking ceramic samples in SBF solution at different times.The obtained results confirmed the bioactivity of this ceramic powder by the apatite formation within a few days.

Figure 1 .
Figure 1.Flowchart of powder synthesis protocol

Figure 3 .
Figure 3. XRD diagrams of ceramic powder before and after soaking in SBF solution

Figure 4 .
Figure 4. FTIR spectra of ceramic powder before and after soaking in SBF solution

Figure 5 .Figure 6 .
Figure 5. SEM images of ceramic powder: a) initial ceramic powder, b) after 1 day, c) after 3 days, d) after 7 days and e) after 15 days of soaking in SBF solution