Characterization of Calcium Aluminate Cement Phases when in Contact with Simulated Body Fluid

An ideal endodontic repairing biomaterial would adhere to the tooth structure, maintain suitable sealing, be insoluble in tissue fluids, be dimensionally stable, non-resorbable, radiopaque and show biocompatibility1. Calcium aluminate-based cement (CAC) has already been studied for biomedical applications as endodontics2-6. This material presented good physical and mechanical properties7, biocompatibility8,9 and acted as a chemical and mechanical barrier against bacterial microleakage4. This sort of cement has also shown an absence of inflammatory reaction and it was biocompatible when tested on rat subcutaneous tissue2. In the presence of radiopacifier (bismuth oxide), it has also presented suitable radiopacity for all studied thicknesses, as recommended by ISO 687610. Another important desired property is bioactivity of endodontic materials, which has been defined as “the bond ability with the host bone tissue”. In order to avoid costly experiments, various in vitro tests have been used to predict the in vivo bone bioactivity of bioceramics. One common method that has been used for testing in vitro bioactivity is to evaluate the apatite-formation ability of bioceramics in the simulated body fluids (SBF)11,12. The examination of apatite formation on the surface of a material in SBF is useful for predicting the in vivo bone bioactivity of the material. Besides that it can reduce both, the number of animals used and the time length of the experiments, speeding up the development of new types of bioactive materials11. For a full evaluation of the in vitro bioactivity it is recommend the combination of SBF and cell experiment methods12,13. The ability to stimulate a cell response is very important and complements the in vitro apatite deposition data. Recent studies involving the use of CAC are based on commercial products comprising a mixture of different crystalline phases2-7. In vitro characterization of commercial CAC has been carried out in simulated body fluid solutions (SBF) prepared according to Kokubo’s (KSBF) or Rigo’s (RSBF) techniques. CAC presented ability of stimulating hydroxyapatite deposition in SBF, although the solution composition defines the sort of phases precipitated. The optimal condition for the calcium phosphate phase precipitation was attained in RSBF and a surface layer of stoichiometric hydroxyapatite could be detected by XRD14. Improvements can be achieved by investigating the CAC production routes aiming at the proper balance between the phases and the control of impurities that may affect its performance for biomedical applications. The production of CAC phases was previously studied by the authors using Al2O3-CaCO3 or Al2O3-CaO as initial raw materials. The Characterization of Calcium Aluminate Cement Phases when in Contact with Simulated Body Fluid


Introduction
An ideal endodontic repairing biomaterial would adhere to the tooth structure, maintain suitable sealing, be insoluble in tissue fluids, be dimensionally stable, non-resorbable, radiopaque and show biocompatibility 1 .
Calcium aluminate-based cement (CAC) has already been studied for biomedical applications as endodontics [2][3][4][5][6] .This material presented good physical and mechanical properties 7 , biocompatibility 8,9 and acted as a chemical and mechanical barrier against bacterial microleakage 4 .This sort of cement has also shown an absence of inflammatory reaction and it was biocompatible when tested on rat subcutaneous tissue 2 .In the presence of radiopacifier (bismuth oxide), it has also presented suitable radiopacity for all studied thicknesses, as recommended by ISO 6876 10 .
Another important desired property is bioactivity of endodontic materials, which has been defined as "the bond ability with the host bone tissue".In order to avoid costly experiments, various in vitro tests have been used to predict the in vivo bone bioactivity of bioceramics.One common method that has been used for testing in vitro bioactivity is to evaluate the apatite-formation ability of bioceramics in the simulated body fluids (SBF) 11,12 .The examination of apatite formation on the surface of a material in SBF is useful for predicting the in vivo bone bioactivity of the material.Besides that it can reduce both, the number of animals used and the time length of the experiments, speeding up the development of new types of bioactive materials 11 .For a full evaluation of the in vitro bioactivity it is recommend the combination of SBF and cell experiment methods 12,13 .The ability to stimulate a cell response is very important and complements the in vitro apatite deposition data.
Recent studies involving the use of CAC are based on commercial products comprising a mixture of different crystalline phases [2][3][4][5][6][7] .In vitro characterization of commercial CAC has been carried out in simulated body fluid solutions (SBF) prepared according to Kokubo's (KSBF) or Rigo's (RSBF) techniques.CAC presented ability of stimulating hydroxyapatite deposition in SBF, although the solution composition defines the sort of phases precipitated.The optimal condition for the calcium phosphate phase precipitation was attained in RSBF and a surface layer of stoichiometric hydroxyapatite could be detected by XRD 14 .
Improvements can be achieved by investigating the CAC production routes aiming at the proper balance between the phases and the control of impurities that may affect its performance for biomedical applications.The production of CAC phases was previously studied by the authors using Al 2 O 3 -CaCO 3 or Al 2 O 3 -CaO as initial raw materials.The Al 2 O 3 -CaO route enabled the production of the target phases (CA, CA 2 , C 3 A and C 12 A 7 ) with higher purity compared to the Al 2 O 3 -CaCO 3 one in which C stands for CaO and A for Al 2 O 3 15 .Therefore, in this work in vitro characterization of calcium aluminate cement phases (CA, CA 2 , C 3 A and C 12 A 7 ) was evaluated when in contact with simulated body fluid solutions.This can highlight the importance of each particular crystalline phase comprising the CAC containing product and provide better tools and insights in terms of designing its composition, application and performance.

Experimental Procedures
Calcium aluminate cement phases were previously synthesized by the CaO and Al 2 O 3 calcination using electrical furnace at different temperatures and dwell time of one hour 15 .
The reactions involved to produce CA, CA 2 , C 3 A and C 12 A 7 phases as listed below: The raw material amounts used and the firing conditions were adjusted in order to produce different CAC phases as presented in Table 1.Following that, the resulting phases were dry ball milled for 17 hours using high alumina spheres with a 5:1 ratio (ball: powder) 15 .
Aqueous suspensions of CAC phases were prepared using a standard laboratory mixer under a 2000 rpm.Deionized water was added to these powders to obtain a homogeneous paste.The solid amount in the suspension is presented in Table 1.C 3 A and C 12 A 7 suspensions were prepared with higher water content.The affinity of CaO with water and the high CaO/Al 2 O 3 ratio in these phases led to heat evolution when in contact with water.Thus, due to water evaporation, it was necessary to increase the content of water added.
The suspensions were used to prepare cylindrical samples (10mm diameter x 5mm height).After shaping, the samples were kept at 37±1 °C in a climatic chamber under a saturate environment (100% R. H.) for 24 hours.After that, the samples were withdrawn out of the molds and kept for 7 days at 37 °C under the same environmental conditions described above.After finishing this step, they were placed into containers with 50 ml of water or SBF solutions and kept at 37 °C (100% R. H.). The pH of these solutions was measured at certain time intervals, using pH electrode connected to an automatic data recorder system.
The SBF solutions named KSBF and RSBF were prepared according to the procedure described in the literature 11,16 , as shown in Table 2.
The 7-day-set samples were also placed into containers with 47 mL of SBF solutions in order to keep a 0.1cm -1 surface area-to-volume ratio and under stirring at 37 °C with the help of a shaker.Afterwards the samples were gently rinsed with deionized water, followed by drying at room temperature according to the literature 17 .The surface of the samples was analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX) before and after treatment in SBF solutions.The samples before and after immersion in SBF were also analyzed by X-ray diffraction (XRD) using CuKα radiation (1.54439 Å) in order to identify the phases formed on the sample surface.

pH tests
CAC phases, mainly CA, C 3 A and C 12 A 7 , increased the water pH (Figure 1A).In simulated body fluid, higher pH values were attained in RSBF.pH measurements with the time for SBF solutions in contact with previously set CA, CA 2 , C 3 A or C 12 A 7 samples showed values in the range 7.5-8.5 as soon as they were placed in KSBF (Figure 1B).Conversely, CA, C 3 A and C 12 A 7 phases presented higher pH in RSBF, reaching values close to 9.5-10 in the first couple of days, whereas pH of approximately 9 was attained for CA 2 one (Figure 1C).

SBF tests
The EDX spectra of the surface of the samples before immersion in SBF confirmed presence of the main chemical constituents of hydrated CA and CA 2 phases (aluminum>oxygen>calcium) (Figure 2A and C) and those for the C 3 A and C 12 A 7 ones (calcium>aluminum>oxygen), as shown in Figures 3A and C.
EDX spectra and SEM analysis of set samples after immersion for 7 days in SBF solutions prepared according to Rigo's are shown in Figures 3 and 4, respectively.The presence of phosphorus was identified mostly for CA 2 indicating the formation of a calcium phosphate phase on the sample surface.For the other phases was identified the presence of Mg 2+ ions on their surfaces (Figures 2 and 3).
XRD analysis of set samples before and after immersion for 7 days in SBF solutions are shown in Figures 5-8 However, the calcium aluminate hydrate formed also depends on the CaO content in the anhydrous phase 18 .For C 3 A and C 12 A 7 as precursors, C 3 AH 6 was the major one (Figures 7 and 8A).On the other hand, for CA and mainly CA 2 , the precipitation of C 2 AH 8 (2CaO.Al 2 O 3 .8H 2 O) and CAH 10 (CaO.Al 2 O 3 .10H 2 O) was identified (Figures 5 and 6A).After hardening and in contact with SBF solution, the hydrates are the main soluble phase, as Al(OH) 3 is insoluble in water.The Ca 2+ ions from the hydrates dissolution react   with HCO 3 -in the solution resulting in calcium carbonate, as identified by XRD (Figures 5-8) according to reactions: The Ca 2+ and OH -ions leached out from the samples, can also react with phosphorus ions of the solution resulting in hydroxyapatite crystals on the material´s surface: According to the literature, the bioactivity has been attributed to the ability to generate hydroxyapatite in the presence of phosphate-containing solution 12,19 .The resulting surface hydroxyapatite layer is similar in structure to the mineral components of bone and teeth 20 .Materials with an apatite surface layer develop a chemical bond and biological integration with the bone 21 .The in vitro precipitation of hydroxyapatite on the surface of a material in SBF indicates Materials Research its bioactivity 17 .However, there is a misunderstanding regarding the concept of bioactivity.Bohner and Lemaitre published a review which questioned whether currently there was enough scientific evidence to support the assumptions around the use of the SBF method 13 .Thus it is recommend the combination of SBF and cell experiment techniques to evaluate the in vitro bioactivity 12 .
Solutions of simulated body fluid used in this work contained similar inorganic ion concentrations to those in human extracellular fluid (human plasma) in order to reproduce the in vitro formation of apatite on bioactive material.SBF is a metastable solution containing calcium and phosphate ions already saturated with apatite 11,16 .The increase in both pH and Ca 2+ concentration result in the supersaturation of SBF solution leading to the precipitation of hydroxyapatite 20 .
However, the OH -ions released are not enough to result in the pH increase and to induce the KSBF supersaturation as its value was kept close to 7.5-8.5 for the whole experiment  time range (Figure 1B).This explains why the formation of a calcium phosphate phase on the sample surface was not detected (Figures 5-8B).
Conversely, the formation of phosphate phases on the CAC phases sample surface was favored in RSBF (Figures 5-8).The main difference between the solutions is the buffering content.According to Rigo et al. 16 , the buffer Tris, (CH 2 OH) 3 CNH 2 , is added only at the right amount to adjust the pH to 7.25, whereas in KSBF, a higher content of this compound is used.This difference could most likely explain the pH increase detected for the RSBF, resulting in the SBF solution supersaturation and consequently the formation of phosphate phases on the sample surface.
The hydrates formed from CA and mainly C 3 A and C 12 A 7 samples are richer in CaO, resulting in more OH -ions leached out from the samples leading to pH values above 9.5 in RSBF, whereas for the CA 2 ones (less rich in CaO) result in lower pH (~ 9.0).
Therefore, for the CA, C 3 A and C 12 A 7 phases in the presence of RSBF the lower content of buffer allowed the faster pH increase (close to 9.5-10).This can be confirmed by the presence of Mg 2+ ions on the surfaces of samples (Figures 2 and 3) which were precipitated from the SBF solution.It has been reported in the literature that magnesium phosphate precipitation is more noticeable at higher pH and inhibits the precipitation of calcium phosphate 22,23 .Thus, calcium carbonate precipitate can be observed by SEM on the CA, C 3 A and C 12 A 7 surfaces (Figure 4).
XRD results also showed that the calcium phosphate was not precipitated on the CA, C 3 A and C 12 A 7 sample surface when in RSBF and that the magnesium ion detected by EDX was most likely precipitated as magnesium phosphate (Figures 5, 7 and 8C).
On the other hand, on the surface of the CA 2 sample after immersion in RSBF, stoichiometric hydroxyapatite [Ca 5 (PO 4 ) 3 OH] was detected (Figure 6C) as the pH condition (approximately 9) did not favor the magnesium phosphate precipitation.Hydroxyapatite is the natural biomineral representing 30 to 70 mass% of bones and teeth 24 .
Besides providing better results for the ability of stimulating hydroxyapatite deposition, Pompeu et al. 25 also indicated the CA 2 phase as the most suitable for applications in endodontics when compared to CA, C 3 A and C 12 A 7 ones.CA 2 presented a lower temperature increase during hydration process, which is essential to avoid necrosis of the bone tissue around the implant region 26 .CA 2 also showed ability to induce an antibacterial environment and interaction with ions in solution simulated body fluid and showed no dissolution in water or SBF.
However, CA 2 presented the longest setting time due to the formation of a more soluble hydrate (CAH 10 ) when compared to a less soluble one, such as C 3 AH 6 .Therefore, to attain a suitable setting time, the addition of accelerating additives is required.The use of cements with a lower setting time should decrease the need of constant professional procedures during the treatment.Besides that, when used as a root-end or root-canal filling material the fast set should also reduce the risk of contamination and dislodgement after placement 27 .

Conclusions
Calcium phosphate phases were not detected on the surface of CAC samples phases after immersion in KSBF as the release of OH -ions was not sufficient to increase the pH and to enhance the KSBF's supersaturation.
Conversely, in RSBF the lower content of buffer allowed a faster pH increase, resulting in the precipitation of magnesium phosphate on CAC sample surfaces.Only for the CA 2 samples, the pH attained favored the calcium phosphate phase precipitation and a surface layer of the stoichiometric hydroxyapatite could be detected by XRD.
Based on its ability of stimulating hydroxyapatite deposition in SBF and other properties, among all phases of calcium aluminate endodontic cement evaluated, the CA 2 one containing accelerating additives can be eligible as the most suitable composition for biomedical purposes.However, in order to evaluate its in vitro bioactivity it is still necessary combination of SBF tests and cell experiments methods.

Figure 1 .
Figure 1.pH as a function of the time for (A) water, (B) SBF (simulated body fluid) Kokubo and (C) SBF Rigo solutions in contact with CA, CA 2 , C 3 A and C 12 A 7 sample phases.

Figure 2 .
Figure 2. EDX spectra of the surface of the phases before immersion in SBF: (A) CA, (C) CA 2 and after 7 days in SBF Rigo: (B) CA RSBF, (D) CA 2 RSBF.

Figure 3 .
Figure 3. EDX spectra of the surface of the phases before immersion in SBF: (A) C 3 A, (C) C 12 A 7 and after 7 days in SBF Rigo: (B) C 3 A RSBF, (D) C 12 A 7 RSBF.

Figure 4 .Figure 5 .
Figure 4. Electron micrographs depicting the surface of the phases (A) CA, (C) C 3 A, (B) CA 2 , and (D) C 12 A 7 after 7 days in SBF Rigo

Figure 6 .
Figure 6.XRD patterns of CA 2 (A) before immersion in SBF; (B) after 7 days in KSBF and (C) after 7 days in RSBF.

Figure 7 .
Figure 7. XRD patterns of C 3 A (A) before immersion in SBF; (B) after 7 days in KSBF and (C) after 7 days in RSBF.

Table 1 .
Processing conditions for the production of calcium aluminate cement phases based on Al 2 O 3 -CaO mixture and the respective solid content in the suspension.
. Calcium phosphate phases were not identified on the surface of CAC phases after treatment with KSBF.On the other hand, magnesium phosphate [Mg 3 (PO 4 ) 2 .22H 2 O] was detected on the surface of CA, C 3 A and C 12 A 7 after immersion in RSBF.Hydroxyapatite [Ca 5 (PO 4 ) 3 OH] was only detected on the surface of CA 2 after immersion in RSBF.The hydration products of commercial CAC (usually comprising a mixture of CA, CA 2 and C 12 A 7 ) in water are calcium aluminate hydrates and aluminum hydroxide.At temperatures above 35 °C, C 3 AH 6 (3CaO.Al 2 O 3 .6H 2 O or Ca 3 [Al(OH) 4 ] 2 (OH) 4 ) is the main hydrated phase 18 :