Si-TCP Synthesized from “Mg-free” Reagents Employed as Calcium Phosphate Cement

The influence of silicon doping on calcium phosphate cement were explored in this work. α -TCP and Si- α -TCP were prepared by solid state reaction employing “Mg-free” CaHPO 4 , CaCO 3 and CaSiO 3 as precursors. It was possible to obtain TCP powders with low contents of β phase as contaminant. Cement liquid phase was an aqueous solution containing 2.5 wt. (%) of Na 2 HPO 4 and 1.5 wt. (%) of citric acid. The liquid-to-powder ratio was 0.6 mL.g –1 . Chemical, physical and mechanical properties of the cement samples were determined by means of XRD, FTIR, XRF, compressive strength and SEM. The calcium phosphate cements obtained achieved satisfactory properties; however, Si- α -TCP presented a decrease on the rate of setting reaction.


Introduction
The need for new biomaterials which could improve life quality of people who suffer from oldness diseases or who have any bone tissue injury due to accidents and diseases like obesity and cancer are resulting in a growing number of researches. In this context, the development of new orthopedics biomaterials based on calcium phosphate compounds is relevant once they present excellent bioactivity and biocompatibility due to its chemical composition similar to the mineral part of bone and teeth [1][2][3] .
Silicon substitution into some phosphorous sites of calcium phosphate bioceramics is a promising approach to develop new biomaterials for orthopedics applications due to the increased bioactivity and cell differentiation on the material's surface which could be promoted by the presence of this element [4][5][6][7][8][9][10][11] . Therefore, silicon doped α-tricalcium phosphate (Si-α-TCP), is attracting the attention of researchers since its employment as bone cement could be of great interest. Nevertheless, it is still not well stabilished if the enhanced biological properties of the silicon doped calcium phosphates compounds is due to the presence of silicon itself or it is because of its influence on the chemical properties of the material [12][13][14] .
Moreover, silicon is known to stabilize the α-tricalcium phosphate, α-TCP, structure and to promote its formation at lower temperatures 15,5,16,17 leading to a cost reduction of its processing. It is well known that the synthesis of a pure α-TCP is not an easy task since all process conditions can change its final properties, or even inhibit its formation. The most limiting factor is the quality of the starting reagents which may preclude the formation of α-TCP at temperatures as high as 1600 °C 10,13 . Therefore, the reproducibility of α-TCP synthesis becomes very difficult and, in some cases, impossible. In a previous work, our group has developed simple synthetic methods to synthesize high purity reagents to eliminate the most important impurity: magnesium, which is an established stabilizer element of β-tricalcium phosphate, β-TCP 16 . It has been discovered that the standardization of the reagents properties guaranteed the reproducibility of α-TCP manufacturing process and the formation of a high purity α-TCP and Si-α-TCP. Thus, the major objective of this study is to investigate the influence of Si on the chemical, physical and mechanical properties of the calcium phosphate cement.

TCP precursors and TCP powders synthesis
"Mg-free" CaHPO 4 and CaCO 3 were synthesized by aqueous solution precipitation in the presence of ethylenediamine tetraacetic acid (EDTA). CaSiO 3 were synthesized by solid state reaction of "Mg-free" CaCO 3 and electronic grade SiO 2 , which was kindly provided by the Photonic Materials Laboratory-UNICAMP, Brazil 16 .
Afterwards, two tricalcium phosphate powders, α-TCP and Si-α-TCP, were synthesized by solid state reaction. The syntheses parameters are displayed in Table 1.

Calcium phosphate cements
Cement samples were prepared using an aqueous solution containing 2.5 wt. (%) of Na 2 HPO 4 and 1.5 wt. (%) of C 6 H 8 O 7 (citric acid) with a liquid-to-powder ratio equal to 0.60 mL.g -1 .
After molding in Teflon molds (6 × 12 mm) samples were left in a 100% relative moisture environment for 24 hours. Then, cement cylinders were polished, demolded and immersed in simulated body fluid (SBF) for 24 and 168 hours at 37 °C. After each immersion period, samples were gently rinsed with distillated water, immersed in acetone to stop the setting reaction and dried at 100 °C for 6 hours.

Characterizations
TCP powders and cement samples crystalline phase analyses were carried out by means of qualitative X ray diffraction (Rigaku DMAX 2200, 20-40° (2θ), 0.01° (2θ)/s, 40 mA and 20 kV). JCPDS files used for phase identification were 09-0348 for α-TCP, 09-0169 for β-TCP and 46-0905 for calcium deficient hydroxyapatite, CDHA. Moreover, a quantitative XRD analysis was performed to quantify β-TCP on the TCP powders. It was employed the internal pattern method in which a diffraction line from the phase being quantified is compared with a diffraction line from a standard mixed with the sample in known proportions 18 . The standard employed was Al 2 O 3 and β-TCP diffraction line used was (2 1 4).
Chemical composition of the samples were evaluated by means of fourier transformed infrared spectroscopy. Samples were diluted in KBr and analyzed in a Perkin Elmer 1600 FT-IR spectrometer with a scanning range from 450 to 4000 cm -1 and resolution of 2 cm -1 .
Powders stoichiometry was determined by quantitative X ray fluorescence (MagiX Super Q Version 3.0 X-ray fluorescence spectrometer, Philips, The Netherlands). Samples were weighed at 0.3000 g, mixed with 5.5 g of spectral grade Li 2 B 4 O 7 and melted in a Pt/Au crucible and formed into disks in a special controlled furnace Perl'X3 (Philips, The Netherlands). Calibration curves were prepared using certified composition standards of natural and synthetic calcium phosphates and calcium silicates. Finally, BET specific surface area and particle size distribution were determined using a Micromeritics ASAP 2010 and a Malvern Mastersizer S, respectively.
Cement setting times were determined using the ASTM-C266-04 standard 19 . Cement samples compressive strength after each time of setting were determined using a MTS, Test Star II with a 10 kN cell attached and a compression velocity of 1 mm/min. The fracture surface was gold coated (BAL-TEC, SCD 050) and its morphology was analyzed on a scanning electron microscope (JEOL, JXA-840A).

Results
For both samples it was possible to obtain TCP powders in its α-TCP form as can be observed on XRD diffractograms of Figure 1. The contents of β-TCP were very low: 8 and 4 wt. (%) for α-TCP and Si-α-TCP, respectively. Powders stoichiometry is displayed on Table 2, Ca/P and Ca/P+Si ratios were 1.50 and 1.46, respectively.  The effectiveness of the solid state reaction employed was also elucidated by FTIR analysis since the absorption bands present on both spectrums ( Figure 2) are characteristic of α-TCP as displayed on Table 3 20 .
Moreover, both TCP powders have very similar BET specific surface area (0.8030 ± 0.0125 and 0.6930 ± 0.0033 m 2 /g for α-TCP and for Si-TCP, respectively) and particle size distribution after 48 hours of ball milling. The mean particle diameter was 9.61 ± 0.14 µm for α-TCP and 10.68 ± 0.08 µm for Si-TCP. This results are resumed on Table 2.
Cement setting times are displayed on Table 4. For both cements the values obtained using the Gilmore Needles 19 were higher when compared to the values reported on the literature 20,21 . Initial setting time was 15 minutes for α-TCP and 30 minutes for Si-α-TCP. Moreover, final setting time was 43 minutes for α-TCP and 120 minutes for Si-α-TCP.
As can be observed on XRD patterns of Figure 1 α-TCP and Si-α-TCP setting reaction occurs by the dissolution of TCP phase and the precipitation of apatite crystals once these are the only crystalline phases observed during the role process. Indeed, as displayed on the FTIR spectrums of Figure 3 the apatite phase formed are calcium deficient hydroxyapatite (CDHA, Ca 9 (HPO 4 )(PO 4 ) 5 OH) since their characteristics absorption bands (Table 5) are present in both spectrums. Moreover, it is possible to verify that both cements lead to an apatite phase which is also carbonated due to the CO 3 2characteristics bands at 850 to 900 and at 1350 to 1600 cm -1 (highlighted with a "*" on Figure 3).
Cement's mechanical strength evolution with time of SBF immersion can be observed on Figure 4. It is verified that Si-α-TCP resulted on lower values of compressive strength after 7 days of immersion. Moreover, during the first day of immersion, Si-α-TCP did not achieve any mechanical resistance while α-TCP achieved values around 5 MPa.    Table 4. Setting times of cement samples. T i = initial setting time and T f = final setting time.

Discussion
The lower content of β-TCP on Si-α-TCP confirms the efficiency of Silicon in stabilizing the α-TCP phase by lowering the β → α phase transformation temperature 4,5,22 once the powder doped with Silicon resulted on purer α-TCP (4 wt. (%) of β-TCP) at a lower sintering temperature work were sintered for longer times it would be expected a reduction on β-TCP content. Samples purity were also confirmed after Ca/P and Ca/P + Si ratios determination since their values are very close to the theoretical ones of TCP (α and β phases) compounds, 1.50.
During cement preparation, it was determined larger values of setting time. In a first moment, this fact can be explained by the high liquid-to-powder ratio employed 0.60 mL.g -1 against 0.32-0.34 mL.g -1 normally used for conventional α-TCP cement 8,20,21,25 ; however, this huge amount of liquid was necessary to guarantee the cement moldability. Moreover, the addition of citric acid to cement's liquid phases has also contributed to the high setting times since this compound increases the TCP particles' wettability and cement paste fluidity caused by a deflocculation on the TCP powder which also leads to a lower rate of setting reaction 26 . It is important to emphasize that without citric acid addition the liquid-to-powder ratio needed was higher than 1.0 mL.g -1 .
Furthermore, by comparing XRD patterns of Figure 1 it is possible to infer that Silicon induces a reduction on the rate of setting reaction. In the first 24 hours, as it was expected, α-TCP (α-TCP-c0) started to solubilize together with CDHA precipitation. Surprisingly, for Si-TCP the setting reaction seems not to occur on the first 48 hours (Si-TCP-c0 and Si-TCP-c1) since only α-TCP diffraction lines are observed on the XRD patterns. Finally, after 168 hours, for α-TCP cement the TCP → CDHA conversion has finished while Si-TCP cement still have some TCP without reacting.
The difference on TCP reactivity is responsible for the lower compressive strength achieved by Si-TCP cement, as displayed on the boxplot chart of Figure 4. At initial times samples "Si-TCP-c0" and "Si-TCP-c1" did not present any mechanical resistance while sample "α-TCP-c0" reached 5.6 ± 0.9 MPa. As TCP → CDHA conversion evolves samples' compressive strength enhances reaching after 168 hours 21.5 ± 2.4 and 14.8 ± 2.6 MPa for α-TCP-c7 and Si-TCP-c7, respectively.
Nevertheless, it is important to observe that even though the mechanical resistance for Si-TCP cement after 168 hour is lower than for α-TCP cement, this material had not reached the 100% TCP → CDHA conversion, thus, it is expected that its maximum mechanical resistance became higher after all Si-TCP is converted into CDHA.

Conclusions
Si-α-TCP was synthesized by a simple solid state reaction in which it was employed "Mg-free" reagents leading to lower sintering temperatures for both Si doped and non-doped α-TCP. Moreover, calcium phosphate cements obtained employing these TCP powders achieved satisfactory properties; however, Silicon has induced a decrease on the setting reaction velocity.  (1250 °C). Based on results reported on the literature [22][23][24] depending on the Magnesium content in the precursors employed the temperature at which pure α-TCP can be synthesized could be raised up to 1500 °C, however, if the material is sintered at the right temperature and for enough time to guarantee the total β → α conversion it is possible to obtain pure α-TCP no matter how high is the Mg contamination. Then, if the powders synthesized on this