Hydration behavior and formation of strätlingite compound (C2ASH8) in a bio-cement based on tri-calcium silicate and mono-calcium aluminate for dental applications: influence of curing medium

The aim of this work was to study the influence of simulated body fluid (SBF) and Pseudomonas aeruginosa (in Mueller Hinton Broth, MHB media) on the hydration reactions, formation of strätlingite (C2ASH8) and morphology of a composite material based on nano-size tri-calcium silicate (C3S) and tri-calcium aluminate (CA) phases. The two experimental phases were prepared in laboratory by firing the required molar ratios of chemically pure reactants by solid-state reactions at elevated temperatures. Investigation of setting time, micro-hardness, pH of immersion solution, X-ray diffraction analysis, infrared spectroscopy, scanning electron microscopy and cytotoxicity were also carried out. The results emphasized that the hydrated compounds of C3S phase can partly solve the problem of conversion process through the formation of the more stable and highly mechanical stratlingite hydrate (C2ASH8) plates that results in an increase in hardness values for samples cured in distilled water (DW) at 37 °C. The SBF solution showed more strength loss than those cured in Pseudomonas aeruginosa microbe (MHB) medium bacterial solution. SEM and EDAX analyses emphasized the formation of hydroxyapatite at 37 °C. The prepared cements are promising for dental application.


Background
Calcium silicate and calcium aluminate cement materials have been successfully improved for use in dentistry. The cements products have many beneficial properties: compressive strength, purity, suitable working and setting times, radiopacity, sealing and film thickness that meet the dental standard. Both cements have antimicrobial action due to their high pH curing media (Huang et al. 2019). They also have biomineralization property that came from the release Ca 2+ and OH − ions when contact with moist tissue. The released ions interact with tissue fluids phosphate ions, thus precipitating calcium phosphate compound (Camilleri 2014;Camilleri and Gandolfi 2010). Beside that both cements are known for their osteogenic property, which is considered an important property for pulpal and periapical tissues wound healing and bone health (Primus et al. 2022).

Open Access
Bulletin of the National Research Centre *Correspondence: smnagi@gmail.com Radwan and Nagi Bulletin of the National Research Centre (2022) 46:180 Calcium aluminate cement (CA) has been considered for over two decades as one of the most common materials used in dental applications as it has suitable setting time and high early crushing strength at room temperature. The superior physico-mechanical properties of calcium aluminate cement during the first 24 h of hydration process makes it promising bio-dental material for different applications in dentistry (Parreiraet al. 2016). Hydration process of CA phase is highly dependent on the temperature of the reaction medium that is at 20 °C, CAH 10 is the predominant hydration product, whereas at temperature higher than 20 °C, the main hydration phases are C 2 AH 8 and AH 3 (Shanget al 2016; Ukrainczyket al 2007; Lee et al. 2001;Bushnell-Watson and Sharp 1986;Taylor 1990). Curing of Calcium aluminate cement in distilled water (DW) at temperature above 35 °C results in the conversion of the metastable CAH 10 and C 2 AH 8 hydrated compounds to the more stable cubic crystals of C 3 AH 6 (hydrogarnet) and AH 3 (gibbsite) in a process called conversion. It was stated that the extent of conversion process increases by increasing the curing temperature and humidity of the curing medium as more metastable phases will dissolve resulting in the formation of the more stable compounds according to the following equations: (Taylor 1990;Luz and Pandolfelli 2012;Bushnell-Watson and Sharp 1986) The conversion process should be avoided as it increases the porosity and micro-cracks of the hardened pastes which will be responsible for the weak physical and mechanical properties (Luz and Pandolfelli 2012;Bushnell-Watson and Sharp 1986). It was found in a previous studies that the presence of free silica and calcium silicate hydrate (CSH) can partly inhibit the conversion process through formation of the more stable strätlingite compound (C 2 ASH 8 ) that has good mechanical (1) CA + 10H → CAH 10 (2) 2CA + 11H → C 2 AH 8 + AH 3 (3) 3CA + 12H → C 3 AH 6 + 2AH 3 (4) 3CAH 10 → C 3 AH 6 + 2AH 3 + 18H (5) 3C 2 AH 8 → 2C 3 AH 6 + AH 3 + 9H properties than hydrogarnet (C 3 AH 6 ) in temperature range of 20-70 °C according to the following equations (Mostafa et al. 2012;Bentsen et al. 1990).
The ultra-pure tri-calcium silicate phase (C 3 S) that is one of the two main calcium silicate phases present in mineral trioxide aggregate (MTA) was one of the cementitious materials used in dentistry (Lee et al. 1993;Lenji et al. 2017;Asgary et al. 2009). It exhibited adequate physico-mechanical properties in addition to good properties needed in dental applications such as good adhesion to tooth, bactericidal properties and bioactivity (Sarkar et al. 2005;Al-Haddad et al. 2017;Accorinte et al. 2008;Asgary and Kamrani 2008). Tri-calcium silicate (C 3 S) phase is a hydraulic cement that can undergo setting and hardening processes in wet curing media such as distilled water (DW), saliva, blood, simulated body fluid (SBF), bacterial media and dentinal fluid through the formation of calcium silicate hydrate (CSH) gel once mixed with water according to the following chemical equation (Lee et al. 1993;Moreno-Vargasa et al. 2017;Camilleri 2007).
Tri-calcium silicate phase (C 3 S) has a relatively prolonged setting time which is not recommended in clinical and dental applications. This may be improved by mixing C 3 S phase with calcium aluminate phase (CA) as it has fast setting and hardening processes (Lee et al. 1993). On the other hand, mixing of C 3 S with CA phase may compensate the conversion process of the main aluminate hydrate compounds (CAH 10 and C 2 AH 8 ) at higher curing temperature by a reactions between CSH hydrate and amorphous free silica (SiO 2 ) present in C 3 S phase powder with CAH 10 and C 2 AH 8 forming the more stable strätlingite compound (C 2 ASH 8 ) (Taylor 1990;Bentsen et al. 1990).
The current research work aims at studying the influence of different curing media, namely, simulated body fluid (SBF) and Pseudomonas aeruginosa (in Mueller Hinton Broth, MHB media) in comparison with distilled water (DW) on the hydration reaction characteristics and morphology of a composite material based on tri-calcium silicate (C 3 S) and tri-calcium aluminate (CA). Investigation of setting time, micro-hardness, pH of immersion solution, X-ray diffraction analysis, infrared spectroscopy, scanning electron microscopy and cytotoxicity of the formulated bio-cement were also carried out.

Materials preparation and characterization
Tri-calcium silicate (C 3 S) phase (17-27 nano-meter) was synthesized by firing molded cubes of 3:1 CaO/ SiO 2 molar ratio using ultra-pure limestone and quartz (99.6% SiO 2 ), in the presence of 0.5% boric acid at 1000 °C for 2-h (Taylor 1990). The product was ground, remolded using carbon tetrachloride and fired at 1450 °C for 2-h. The firing process was repeated until completion of the reaction. The end product was checked for the presence of free lime. The mono-calcium aluminate (CA) phase (73-113 nano-meter) was prepared in laboratory by dry mixing 1:1 molar ratio of CaCO 3 (≈99.8%) and Al 2 O 3 (99.6% Alumina). The required 2-inch cubes were pressed from the homogenized molar mixture to be ready for firing at 1000 °C for 2 h, and then the cooled material was ground and remolded with the aid of carbon tetrachloride and fired at 1500 °C for 6 h. The final phase agreed with some studies that emphasize the levels of C 12 A 7 and CA 2 decreased while CA phase increased with temperature and time (Taylor 1990;Emaraet al 2018;Rivas Mercuryet al 2005). The synthesized materials were ground for 15 h in an agate mill until the desired grain size. There were investigated by X-ray diffraction to characterize the synthesized phase (Fig. 1). The particle diameter in nano-meter was determined with the aid of transmission electron microscope (JEM-1230) at 100 kV to evaluate the particle size (Fig. 2). In this investigation, a composite of bio-cement was formulated from the prepared tri-calcium silicate (C 3 S) and mono-calcium aluminate (CA) phases according to the following weight percentages: • 40 wt% C 3 S phase.
The homogenized dry composite material was finely ground for 3 h using Retsch GmbH PM100, Germany milling machine. One day hardened pastes were cured in incubator at 37 °C in the three curing media that are distilled water (DW), simulated body fluid (SBF) and Pseudomonas aeruginosa in Mueller Hinton Broth (MHB media). Preparation of a workable paste was done at a water/powder ratio of 0.23 ml/g. The paste sample was prepared using cylindrical mold of dimensions: diameter = 10 mm and height = 2 mm. It was poured in the mold in two equal layers, each layer was compacted and pressed in a homogenous specimen. After molding, the samples were cured in 100% humidity chamber at a constant temperature of 37 °C for 24 h. The samples were de-molded and cured in the different media at 37 °C until testing at 3, 7 and 14 days. • Preparation of SBF solution was done in laboratory according to Kokubo et al. (1990). The appropriate ratios of chemically pure NaCl, NaHCO 3 , KCl, K 2 HPO 4 .3H 2 O, MgCl 2 .6H 2 O, CaCl 2 and Na 2 SO 4 were dissolved in deionized water at pH = 7.4 with the aid of Tris-(hydroxyl methyl)-amino methane [(CH 2 OH) 3 CNH 3 ] and hydrochloric acid buffer solution as shown in Table 1. • Incubation of the material was carried out in Mueller Hinton Broth media (Eliopoulus et al. 1989); 25 ml of the broth in 100 ml conical flasks was first sterilized and inoculated with 200 µl of Pseudomonas aeruginosa at 3.5 × 100 CFU/ml. • The chemical composition of the media (Gram/Liter) is given as follows (Kokubo et al. 1990): • Beef infusion solids 2.0 • Starch 1.5 • Casein hydrolysate 17.0 • pH after sterilization 7.4 ± 0.1 • Temp. 25 °C

Setting time
The final setting time of the formulated bio-composite was carried out using Vicat apparatus using the past prepared with distilled water for 30 s at a water/powder ratio of 0.23 ml/g. The paste was poured into a disk mold of 10 mm diameter and 2 mm in thickness. After 120 s from the start mixing time, a Gilmore needle (with a flat end diameter of 2 mm and weighting 100 g load) was gently lowered to the surface of the tested sample. This procedure was repeated at 30-s intervals until the indenter failed to make a complete circular mark on the tested material. The recorded average setting time of set of five disk samples prepared from the synthesized composite bio-material was taken.

X-ray diffraction
The X-ray diffraction analysis was carried out on some selected hydrated samples with the aid of Philips X-ray diffractometer PW 1730 with Ni-filtered Cu-Kα X-ray radiation (λ = 1.5406 Å) powered at 40 kV and 30 mA.
Diffraction data were recorded in the 2θ range from 5° to 70°, counting for 10 s in steps of Δ(2θ) = 0.01°.

ATR/FTIR spectroscopy
IR spectra were recorded on some selected samples with the aid of JASCO model FTIR 6100 spectrometer made in Japan in the range 4000-400 cm −1 with resolution 4 cm −1 .

SEM-EDS analysis
Investigation of the morphology of some selected cured samples was done by scanning electron microscopy (SEM) together with energy-dispersive X-ray spectra (EDX) using an instrument (type Inspect S, T810, D8571, FEI Co., Japan) with an accelerating voltage of 30 kV and a magnification from 10× to 300,000×.

pH of the immersion solution
Disk specimens of cement paste were prepared by mixing with distilled water for 30 s at a water/powder ratio of 0.25 ml/g. The workable mix was then placed in a mold that measured 10 mm diameter and 2 mm in thickness. Then, the prepared disks were directly placed in 20 ml of each curing medium mentioned above (Eliopoulus et al. 1989;Nurit et al. 1993) (and kept at 37 °C in a 100% humidity water bath for 3-, 7-and 14-day curing periods. The pH values of the immersion solution were determined using a solid-state pH sensor connected to a pH meter (Medika Scientific Jenway bench top pH meter, England).

Determination of calcium and phosphorus ions
The concentration of calcium and phosphorus ions released in the curing media were measured by Agilent 5100 Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES) with Synchronous Vertical Dual View (SVDV). For each series of measurements intensity calibration curve was constructed composed of a blank and three or more standards from Merck Company (Germany). Accuracy and precision of the measurements were confirmed using external reference standards from Merck, and standard reference material for trance elements in water and quality control sample from National Institute of Standards and Technology (NIST) were used to confirm the instrument reading (Wang et al. 2008).

Micro-hardness
Micro-hardness testing was performed at two different time intervals (24 h and 28 days). Ten specimens of 15 mm diameter and 2 mm thick were prepared using distilled water (DW) and allowed to cure for 24 h at 37 ± 1 °C in an incubator. The specimens were then removed from the molds and subjected for micro-hardness test (24 h) using Vickers micro-hardness tester (Nexus 4503, Innova Test, Netherlands, Europe). Three randomized indentations were made with a 100-g load, with a dwell time of 10 s. For randomization, specimens were arbitrarily rotated before indentations. Then, the tested specimens were randomly divided into two groups according to the immersion media; either immersed in 20 ml of each of DW, SBF and bacterial solution for 28 days. After immersion in the three curing media for 3, 7 and 28 days, the specimens were dried in a desiccator. The surface of each disk was ground using progressively finer grits of silicon carbide paper, from 180 to 1200 grit, and finally polished using 3-micron polycrystalline diamond paste. Then measurement of micro-hardness was performed again as mentioned before (APHA et al. 2017).

Cytotoxicity test Cell culture
• Preparation of sample.
Black plastic sample was soaked in alone with media for 48 h in refrigerator, and then the media was added to the cell.
Cells were batch cultured for 5 days and then seeded at concentration of 10 × 103 cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37 °C for 24 h under 5% CO 2 using a water jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added, and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration of (ug/ml). After 48 h of incubation, medium was aspirated, and 40 ul MTT salt (2.5 μg/ml) was added to each well and incubated for further 4 h at 37 °C under 5% CO 2 . To stop the reaction and dissolving the formed crystals, 200 μL of 10% sodium dodecyl sulfate (SDS) in deionized water was added to each well and incubated overnight at 37 °C. A positive control which composed of 100 µg/ml was used as a known cytotoxic natural agent who gives 100% lethality under the same conditions. The absorbance was then measured using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t test by SPSS 11 program. DMSO is the vehicle used for dissolution of plant extracts and its final concentration on the cells was less than 0.2%. The percentage of change in viability was calculated according to the formula: ((Reading of extract/ Reading of negative control) − 1) × 100.
A probit analysis was carried out for IC50 and IC90 determination using SPSS 11 program (APHA et al. 2017).

Setting time and microhardness
Evaluation of the final setting time was carried out on the material pastes of normal consistency prepared with distilled water at a liquid/solid ratio of 0.23 ml/g as given in "Setting time" section. The recorded average setting time was 20 min at 37 °C (average of five readings). Figure 3 shows the hardness data of samples cured in the different hydration media at all curing ages. Figure 3 indicates that the hardness values of samples cured in distilled water (DW) increase with the hydration period from one day and up to 14 days, while for samples cured in SBF solution and the Pseudomonas aeruginosa microbe in MHB media there was a decrease in the hardness values at all curing ages. However, the samples immersed in the microbe medium showed slightly higher hardness values than those cured in SBF solution at all hydration periods.

pH of the curing media
The pH values of the curing media as a function of immersing ages are illustrated in Fig. 4. The pH data reveal that there is a little increase in the pH values of the three immersion liquids with the hydration period.
As can be seen from Fig. 4, the samples cured in distilled water DW showed the highest pH values, while the lowest pH values were recorded for the samples cured in SBF solution.   Fig. 6. The data indicate a phosphorus ions decrease from 3-to 7-day samples followed with no change for the 14-day sample.

X-ray diffraction
Figures 7 and 8 represent the X-ray diffraction patterns of 3-and 14-day samples cured in distilled water (DW) in comparison with those cured in both SBF solution and Pseudomonas aeruginosa microbe medium. The characteristic peaks of the remaining anhydrous C 3 S and CA particles could be detected in the XRD patterns shown in Figs. 7 and 8. Due to the complex hydration system of the formulated bio-composite, the overlapping of the some characteristics XRD peaks could be detected in all patterns. Investigation of Figs. 7 and 8 showed a decrease in the peaks heights of the anhydrous C 3 S and CA phases for samples cured in the microbe MHB medium more than those cured in SBF solution comparing to the sample immersed in distilled water (DW). The XRD peaks of the main hydration products of calcium aluminate phase (CAH 10 and C 2 AH 8 ) were found in the patterns separate or overlapping with other peaks and they showed a slight peak height increase for the sample stored in microbe solution than for those cured in SBF solution for both 3and 14-day samples. The X-ray diffraction peaks of gibbsite (AH 3 ) and hydrogarnet (CAH 6 ) are clearly detected, due to the conversion process of the main hydrated phase, CAH 10 and C 2 AH 8 in addition to those of strätlingite compound (C 2 ASH 8 ) overlapped with other XRD peaks that is formed due to the expected reaction of the main hydrated compounds with the free silica present in C 3 S phase and the calcium silicate hydrate (C-S-H) at 37 °C. They showed a lower peaks heights for the samples cured in the SBF solution. The X-ray diffraction peaks of calcium silicate hydrate (CSH) that is the hydrated phase of C 3 S could be seen in all patterns but overlapping with other XRD peaks. The free lime [Ca(OH) 2 ] liberated during the hydration reactions of C 3 S phase showed its XRD peak more clear for samples cured under distilled water (DW) with a minimum height for the sample cured in the MHB media. The XRD peaks characteristic for hydroxyapatite (HA) that may be formed on the surface of the hardened material immersed in SBF solution could be detected in the XRD pattern of samples cured in the SBF medium but overlapped with those XRD peaks characteristic for the phase in the same d-spacing making an increase in their heights as shown in Figs. 7 and 8.

FTIR spectroscopy
Figures 9 and 10 illustrate the FTIR spectral bands of the 3-and 14-day samples cured in SBF solution and the microbe in (MHB) media in comparison with those hydrated in distilled water (DW). Due to the complex hydration process of the synthesized bio-composite material which results in a many hydration compounds and byproducts that may cause overlapping of their IR bands, so, it was better to collect the results of FTIR analysis in Table 2. Investigation of the IR results illustrated in Table 2 and Figs. 9 and 10 showed the presence of the IR bands corresponding to the calcium silicate hydrate (CSH) at ≈ 445-815-950 cm −1 overlapping with those at ≈ 491-667 cm −1 for Si-O-Si bond which showed a maximum intensities for the sample cured in distilled water (DW) followed by those immersed in the microbe medium (MHB) and their minimum for the SBF solution. The main hydrated phases of calcium aluminate phase (CA) that are CAH 10 , C 2 AH 8 and C 3 AH 6 showed their characteristic IR bands of their water molecules of hydration at ≈ 524-3465-3670 cm −1 . The bands corresponding to the vibration of C 3 AH 6 , Al-O 6 and Al-O are overlapping in the same transmittance wave length region of ≈ 500-600 cm −1 and at ≈ 800-850 cm −1 for Al-O vibration. The H-O-H bending vibration band of CAH 10 was found at ≈ 1600-1650 cm −1 . The O-H bending vibration for the gibbsite (AH 3 ) can be seen at ≈ 970-1027 cm −1 and those attributed to the O-H stretching vibration also for the gibbsite is located at ≈ 3475-3630 cm −1 . The IR band at ≈ 1000-1100 cm −1 is attributed to α-Al(OH) 3 . The IR bands characteristic for symmetric stretching vibration ύ 3 PO 4 3− corresponding to the hydroxyapatite (HA) are overlapped in the region of 1050-1100 cm −1 and those corresponding to the bending vibration of PO 4 3− at the range of 560-650 cm −1 . The IR spectral data of the 14-day samples showed in Fig. 10 showed an increase in the bands' intensities that may be due to the formation of hydroxyapatite (HA) on the surface of the hardened paste sample. The characteristic IR bands of anti-symmetric stretching ύ 2 and ύ 3 CO 3 2− ~ 850, 1400-1500 cm −1 were found all samples immersed in the bacterial solution, but they showed a very small shoulders for the one and 14-day samples of distilled water. The IR bands at ≈ 3648 and the broad band at ≈ 2500-3700 are mainly attributed to OH − and hydration water (H 2 O) molecules. Figure 11 illustrates the morphology features of the 14-day samples cured in distilled water (DW), simulated body fluid solution (SBF) and Pseudomonas aeruginosa microbe (MHB) medium. As can be seen from the SEM micrographs shown in Fig. 11, there is a clear difference in the microstructures of the three tested samples. The paste sample cured in SBF solution (Fig. 11, plate b) showed a more porous and weak microstructure than the other two samples. The sample  (Fig. 11, plate a) showed the highest dense and closed texture than the other hardened samples (plates b and c). The SEM micrographs shown in Fig. 11 showed these mi-crystalline fibrous of calcium silicate hydrate (CSH) gel covering the anhydrous C 3 S grains and other hydrated compounds. A little amount of Ca(OH) 2 plates embedded in pores and between the complex hydrated phases. Investigation of the micrographs (plates a-c) shown in Fig. 11 emphasized the presence of the hexagonal crystals of the main hydration products of calcium aluminate phase (CA) that are CAH 10 and C 2 AH 8 in addition to the cubic crystals of hydrogarnet (C 3 AH 6 ) and aluminum hydroxide (AH 3 ) gel formed due to the conversion process of the main hydrated phases (CAH 10 and C 2 AH 8 ) at 37 °C. The microstructure of sample cured in DW (Fig. 11, plate a) reveals the presence of stratlingite hydrates (C 2 ASH 8 ) plates more than the samples cured in SBF and bacterial solutions together with a considerable amount of amorphous AH 3 gel and thin fibrous particles of C-S-H. the A mesh-like platelets of hydroxyapatite (HA) were detected in the micrograph shown in Fig. 11, plate b that are precipitated from the SBF solution on the surface of both anhydrous and hydrated grains. The micrograph of the sample cured under the microbe medium, plate c, showed a huge amounts of the Pseudomonas aeruginosa microbe embedded between the hydrated compounds and inside the microporous system. EDAX analysis of 14-day sample cured in distilled water (DW), simulated body fluid (SBF) and Pseudomonas aeruginosa in Mueller Hinton Broth, respectively, is shown in Figs. 12, 13 and 14.

Cytotoxicity test
The sample was tested against the normal human epithelial cell line: -1 BJ1 (normal skin fibroblast with sample concentration range between 100 and 0.78 µg/ml) using MTT assay. The cytotoxicity data showed that the synthesized composite was highly safe to normal skin fibroblast cells with cell variability of 93.2% at 100 ppm sample concentration.

Discussion
The hydration process of the formulated bio-cement composite (1:1 C 3 S and CA) proceeded immediately after mixing the dry powder with the appropriate W/S ratio (0.23) of distilled water (DW) according to the above-mentioned chemical equations (Eqs. 1, 2, 6) that is resulting in precipitation of the main hydrated compounds of CA phase (CAH 10 and C 2 AH 8 ) and calcium silicate hydrate (CSH) during the first few minutes of the hydration process to get the finally set samples at average setting time of 20 min that is considered acceptable for medical and dental applications. Calcium aluminate cement has an important role in improving the setting and hardening of the formulated material as it gains about 80% of its hydration reactions during the first 24 h of hydration process with DW, whereas tri-calcium silicate phase needs relatively longer setting and hardening time to get ≈ 80% of its hydration reactions (Bushnell-Watson and Sharp 1990;Taylor 1990). As was mentioned above in the experimental part, curing of 24 h pastes was carried out under different media in an incubator at 37 °C which will have an acceleration effect on the hydration reactions of C 3 S phase resulting in more calcium silicate hydrate and calcium hydroxide [Ca(OH) 2 ] are being precipitated during the hardening process (Bushnell-Watson and Shrap 1990;Taylor 1990). Mono-calcium aluminate phase (CA) is considered the essential aluminate phase which is responsible for the strength development. The main hydration compounds are CAH 10 at 5-10 °C, C 2 AH 8 and AH 3 at 22-35 °C and C 3 AH 6 and AH 3 at a temperature over 35 °C. The conversion process of the calcium aluminate hydrate compounds is highly dependent on temperature and curing periods in which all hydration products will be converted to the cubic hydrogarnet (C 3 AH 6 ) and gibbsite (AH 3 ).  The weakening of mechanical properties in hot and humid curing conditions is mainly due to the conversion of the main hydrated phases, CAH 10 and C 2 AH 8 , (hexagonal crystals) to the more stable cubic hydrogarnet crystals according to Eqs. (4) and (5) (Taylor 1990;Bassyouni et al. 2014). Tri-calcium silicate (C 3 S) phase is mainly responsible for the hardening and strength development of the prepared pastes at later hydration periods through the formation of calcium silicate hydrate (CSH) according to Eq. (6). It was also found that the presence of free amorphous silica (SiO 2 ) in the composition of C 3 S phase together with the calcium silicate hydrate due to the hydration of C 3 S phase have an important role in decreasing the adverse effect of conversion process on the mechanical properties of the hardened pastes at higher curing temperature (37 °C) through forming the more stable strätlingite compound (C 2 ASH 8 ) according to Eqs. (7)-(12) (Taylor 1990;Bassyouni et al. 2014;Matrusinovic et al. 2003): In order to provide adequate radio opacity, bismuth oxide was added to the composition of the synthesized material as it has no adverse effect on the hydration of the two phases used in this study. Due to the fact that hydration reactions of both C 3 S and CA phases are very sensitive to the ions present in the curing medium, so, in this investigation the influence of curing medium namely simulated body fluid (SBF) and Pseudomonas aeruginosa in Mueller Hinton Broth (MHB media) in comparison with distilled water (DW) on the hydration reactions and hardening process of the prepared material was studied.
The hardness Vickers values (Fig. 3) of paste samples cured under distilled water (DW) at 37 °C showed a steady increase with curing age from 1 and up to 14 days due to formation of the main hydrated products of both C 3 S and CA phases according to Eqs. (1), (2) and (6) (CSH, CAH 10 and C 2 AH 8 ) that are responsible for the strength development (Taylor 1990). The conversion (7) 2CAH 10 of CAH 10 and C 2 AH 8 , (hexagonal crystals) to the more stable cubic hydrogarnet crystals (C 3 AH 6 ) at 37 °C curing temperature was diminished by the reactions of free amorphous silica (SiO 2 ) and CSH with aluminate hydrates to give the hydraulic strätlingite compound (C 2 ASH 8 ) of good mechanical properties according to chemical equations (Eqs. 7-12). It was found that strätlingite (C 2 ASH 8 ) may improve the mechanical properties of CA pastes cured in hot and humid conditions as it is precipitated in the pores formed due to the conversion reactions of the metastable hexagonal crystals of CAH 10 and C 2 AH 8 that are responsible for physico-mechanical properties to the more stable C 3 AH 6 (cubic crystals) and AH 3 gel that have an adverse effect on density and hardness (Scrivener and Capmas 1998;Edmonds and Majumdar 1989;Fentiman and Rashid 1990;Matrusinovic et al. 2003). The SEM micrograph of 14-day sample cured in DW represented in Fig. 10, plate a clearly showed plates of strätlingite compound (C 2 ASH 8 ) in a highly closed and dense microstructure than the sample cured in SBF and bacterial solutions. On the other hand, the data shown in Fig. 3 illustrate that the hardness values of samples cured in SBF and bacterial media slightly decreased with the curing age, whereas sample cured in bacterial medium showed slightly higher values than those cured in SBF solution. The decrease in hardness values in case of SBF and bacterial media is mainly due to the conversion process that is very critical during later hydration ages as it adversely affect the physico-mechanical and morphological characteristics of CA phase. The conversion process results in a highly porous structure and poor mechanical properties of the hardened pastes which will be solved by tri-calcium silicate phase (C 3 S) present in the synthesized material through the formation of strätlingite compound (C 2 ASH 8 ) that is precipitated in this porous system leading to an improved hardness values for samples cured in distilled water (Fig. 3) according to chemical . The composition of SBF solution (Table 1) is considered a highly ionized curing medium because it composed of a number of soluble salts, namely, NaCl, NaHCO 3 , KCl, K 2 HPO 4 .3H 2 O, MgCl 2 .6H 2 O, CaCl 2 and Na 2 SO 4 that are completely soluble in distilled water and there will be various free radicals, some of these free radical may act as an accelerator for the hydration reactions of C 3 S and CA phases, while the others are considered aggressive salts for C 3 S phase only because calcium aluminate phase is highly resistant to these aggressive salts (Taylor 1990). Due to the fact that hydration processes of C 3 S and CA phases are very sensitive to some cations and anions (Na + , K + , Ca ++ , OH − and Cl − ), these free radicals may enhance the hydration reactions of both phases as they will act as a nucleating center for the hydrophilic Page 12 of 14 Radwan and Nagi Bulletin of the National Research Centre (2022) 46:180 particles that pushing the hydration reactions to completion (Bushnell-Watson and Sharp 1986;Rivas Mercury et al. 2005;Shang et al. 2016 (Taylor 1990).
The above-mentioned dual adverse effects on the mechanical properties of pastes cured in SBF solution may emphasize the continuous decrease in the hardness values more than those cured in bacterial solution as the Pseudomonas aeruginosa bacteria which may cause only softening of sample surface by the bacterial attack on the hydrated silicate and aluminate compounds without affecting the reaction between CSH and amorphous silica with the hydrated calcium aluminate compounds to form strätlingite compound (C 2 ASH 8 ) which is emphasized by the presence of its plates in the micrograph of samples cured in bacterial solution (Fig. 10, plate c). The higher pH values (alkalinity) of the hydration media are mainly attributed to the liberated calcium hydroxide [Ca(OH) 2 ] from the hydration of C 3 S phase (Eq. 6). The pH data illustrated in Fig. 4 indicate higher pH values for samples stored in distilled water (DW) than those cured in SBF and bacterial solutions at all curing ages that is in agreement with the hardness data in that the hydration reactions of the synthesized material were proceeded normally including the formation of strätlingite compound (Eqs. 7-12) which is responsible for the improvement in mechanical properties. The increased pH values of SBF solution than bacterial medium may be (13) 4Mg(OH) 2 + SiO 2 gel → 4MgO.SiO 2 .8.5H 2 O attributed to the partial dissolution of calcium aluminate compounds in the hydration medium due to the effect of cations and anions present in SBF solution (Table 1) (Taylor 1990;Bassyouni et al. 2014). Figure 5 emphasizes the highest Ca 2+ ion values in SBF hydration medium at all curing ages due to the dissolution of both calcium silicate and calcium aluminate compounds as was discussed above, while the bacterial solution showed the lowest Ca 2+ ion values. For the samples cured in SBF solution, there will be a great tendency for the precipitation of hydroxyapatite (HA) on the surface of the hardened pastes. The values of phosphorus ion concentration of SBF medium shown in Fig. 6 showed a decrease from 3-to 7-day samples due to the reaction of the phosphate ions present in the composition of SBF solution in the free Ca 2+ to form hydroxyapatite (HA) on the surface of calcium aluminate hydrate and calcium silicate hydrate that emphasized the biocompatibility of the formulated material (Iftekhar et al. 2008).
The findings of XRD analysis for the 3-and 14-day samples shown in Figs. 7 and 8 are in agreement with the hardness data as the samples cured in distilled water (DW) showed the maximum decrease in the XRD peak intensities of anhydrous particles of both C 3 S and CA phases that means an increasing extent of the hydration reactions more than the SBF and bacterial media. The peak intensities characteristic of the calcium aluminate and calcium silicate hydrates were higher for samples cured in DW followed by those stored in bacterial medium and the lowest intensities was recorded for the samples in SBF solution. The characteristic XRD peak of the strätlingite compound (C 2 ASH 8 ) that is formed during the conversion reactions of CA phase according to Eqs. (7)-(12) could be found in the XRD patterns of both 3-and 7-day sample in DW more than samples stored in SBF and bacterial solutions. The XRD pattern of the samples cured in SBF solution emphasized the formation of hydroxyapatite (HA) with a slightly increased intensities for 14 days of curing age. The FTIR spectral analysis (Figs. 9,10) are in agreement with the XRD results concerning the IR bands for the main hydrated phase of the formulated material and increased bands intensities for the 3-and 14-day samples in DW more than those stored in SBF and bacterial solutions. Also, the characteristic IR bands of anti-symmetric stretching ύ 2 and ύ 3 CO 3 2− ~ 850, 1400-1500 cm −1 were found for all samples due to the carbonation of the hydrated compounds by atmospheric CO 2 . Formation of hydroxyapatite (HA) for all samples cured in SBF solution was emphasized by detection of the IR bands characteristic for symmetric stretching vibration ύ 3 PO 4 3− overlapped in the region of 1050-1100 cm −1 and those corresponding Page 13 of 14 Radwan and Nagi Bulletin of the National Research Centre (2022) 46:180 to the bending vibration of PO 4 3− at the range of 560-650 cm −1 . The morphology changes of the 14-day samples as influenced by the curing media are illustrated in Fig. 11. The samples cured in DW (plate a) showed the most dense and closed microstructure including the main hydrated compounds of C 3 S and CA phases, namely, fibrous of CSH gel, hexagonal crystals of CAH 10 and C 2 AH 8 in addition to the cubic crystals of hydrogarnet (C 3 AH 6 ) and aluminum hydroxide (AH 3 ) gel. The microstructure of samples stored in simulated body fluid solution (SBF) showed more porous structure than those cured in Pseudomonas aeruginosa microbe (MHB) medium that is in agreement with the above-mentioned findings of hardness values due to the conversion reactions in which the cubic crystals of hydrogarnet (C 3 AH 6 ) and aluminum hydroxide (AH 3 ) gel that were formed at 37 °C. The stratlingite hydrate (C 2 ASH 8 ) plates were clearly found in samples cured in DW as well as some of this plates could be also detected in pastes cured in the bacterial medium, whereas for samples stored in SBF solution it almost not found.

Conclusions
Mixing of mono-calcium aluminate phase with ultrapure tri-calcium silicate phase in ratio of 1:1 by weight improved the setting and hardening processes during the first day of hydration after mixing with distilled water. Curing of mono-calcium aluminate phase in the incubator at 37 °C results in a process called conversion in which all hydration products will be converted to the cubic hydrogarnet (C 3 AH 6 ) and gibbsite (AH 3 ) in a more porous and mechanically weak structure. The results emphasized that the hydrated compounds of C 3 S phase can partly solve the problem of conversion process through the formation of the more stable and highly mechanical stratlingite hydrate (C 2 ASH 8 ) plates which was clearly detected in micrograph of samples cured in distilled water and good hardness values at all hydration ages. The morphology and mechanical properties of samples cured in simulated body fluid (SBF) have adversely affected as the cations and anions present in SBF solution result in an acceleration, aggressive and dissolution effects on the anhydrous particles and hydrated compounds of both C 3 S and CA phases. The samples cured in Pseudomonas aeruginosa microbe (MHB) medium exhibited better mechanical properties and morphology features than those cured in SBF solution with a considerable amount of stratlingite hydrate (C 2 ASH 8 ) plates embedded in the highly porous structure due to the conversion reactions of CA phases. For samples cured in SBF solution, the precipitation of hydroxyapatite (HA) on the surfaces of anhydrous and hydrated particles as well as inside the more porous system of pastes was emphasized by XRD, EDAX and SEM analyses.
Thus, it could be concluded that the prepared cements are promising for different dental application fields.

Recommendations
In vitro followed by in vivo studies for the application of these cements in different dental applications should be performed.