Poorly crystallized hydroxyapatite and calcium silicate hydrate composites: Synthesis, characterization and soaking in simulated body fluid

Materials Characterization 161 (2020) 110158

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Poorly crystallized hydroxyapatite and calcium silicate hydrate composites: Synthesis, characterization and soaking in simulated body fluid

T

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Anna P. Solonenkoa, , Alexander I. Blesmanb,c, Denis A. Polonyankinb,c a

Scientific laboratory, Stomatological Department, Omsk State Medical University, 644099 Omsk, Russia Physics Department, Radio Engineering Faculty, Omsk State Technical University, 644050 Omsk, Russia c Scientific-educational Resource Centre “Nanotechnology”, Omsk State Technical University, 644050 Omsk, Russia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Chemical precipitation method Phase characterization Chemical composition In vitro resorbtion Apatite spherule Biomaterial

Development of the composites based on substances with a high bioactivity potential is of current interest to medical materials science because composites' properties can be modified by varying the components ratios. Weakly crystallized calcium phosphates and calcium silicate hydrates, possessing higher chemical reactivity than their crystalline forms, are promising salts to be studied as biomaterials' constituents. In this study, precipitation from aqueous solutions was employed to obtain apatite and calcium silicate hydrate composites. Synthesized solids were examined without high-temperature thermal pretreatment using X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), laser diffraction, physical adsorption (BET method) and thermogravimetric analysis coupled with the mass spectrometry (TGA/MS). Compositions containing poorly crystallized carbonate-substituted hydroxyapatite, calcium silicate hydrate and calcite as an admixture were obtained by varying concentrations of the reagents. Soaking of the composite ceramics formed from freshly precipitated synthetic solids in simulated body fluid (SBF) at 37 °C for 14 days lead to formation of the surface layer of amorphous calcium phosphate. As the calcium silicate content in the composites increases, the coating's density and thickness also increase, because silicate ions act as active sites in the process of new phase nucleation. Furthermore, prismatic calcite crystals were identified to form on the lower side of ceramics. This may be caused by an increasing of local supersaturation with respect to calcium carbonate in small-volume confinement conditions during the experiment.

1. Introduction One of the current trends in biomaterial science is designing of the composites consisting of the components with different composition. Potentially, optimal properties of the resulting materials can be reached by varying component combinations and ratios. Accordingly, in order to improve bioactivity and resorption rate of the materials for bone defects' reparation, there have been numerous attempts to design solid mixtures of calcium phosphates (CP) with various stoichiometric ratios or composites based on CP and other biocompatible inorganic salts. Currently, mixtures of Са10(РО4)6(ОН)2 (hydroxyapatite, HA) and (α-/ β-)Са3(РО4)2 (tricalcium phosphate, TCP) are the most thoroughly studied and commercially available [1,2]. Also reported are the materials consisting of Ca10(РО4)6(ОН)2 and CaНРО4·2Н2О [3], Ca10(РО4)6(ОН)2 and CaНРО4 [4], Ca10(РО4)6(ОН)2 and Са8Н2(РО4)6∙5Н2О [5], β-Ca3(РО4)2 and Ca2Р2О7 [6], Са4(РО4)2О and

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α-Ca3(РО4)2 [7], β-Ca3(РО4)2 and α-Ca3(РО4)2 [8], Ca10(РО4)6(ОН)2 and CaCО3 [9,10], Ca10(PO4)6(OH)2 and CaSO4 [11], Ca10(PO4)6(OH)2 and Ca18Mg2H2(PO4)14 [12]. In addition to the above-mentioned mixtures of salts, calcium silicates (CS) can be considered as promising CP dopants. Due to higher solubility of CS (in comparison with HA and TCP) and known partaking of SiO32− ions in the skeletal system's reparative processes [13], the addition of CS may affect the bioactivity and resorbability of calcium phosphate containing materials. In this regard, studies are conducted to synthesize CP and CS composites and determine their properties. Results of studies that aimed to obtain mixtures of Ca10(PO4)6(OH)2 or β-Ca3(PO4)2 with β-CaSiO3 (wollastonite, WT) have been previously reported in [14–27]. Composites with wide range of HA (or CS) content (from 10 to 90 wt% [22]) were obtained by mechanical mixing [14–17], sol-gel synthesis [18,19], co-precipitation [20,21], and HA (or CS) crystallization in the presence of commercially available β-CaSiO3

Corresponding author at: 12 Lenina Ave, 644099 Omsk, Russia. E-mail address: [email protected] (A.P. Solonenko).

https://doi.org/10.1016/j.matchar.2020.110158 Received 25 July 2019; Received in revised form 18 January 2020; Accepted 21 January 2020 Available online 24 January 2020 1044-5803/ © 2020 Elsevier Inc. All rights reserved.

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(or β-Ca3(PO4)2, respectively) in the reaction medium [22–25]. In vitro and in vivo studies of biochemical properties of the synthetic materials showed that the dissolution rate of composite samples increased with the increase of CS content [22], and that the interaction of composite ceramics with an SBF produces a bone-like apatite layer on their surfaces which thickens with the increase of wollastonite ratio [25]. CP/CS based materials had an excellent osteoconductivity and could stimulate rapid bone tissue formation [17]. Moreover, the composites were capable to improve a rat bone marrow-derived mesenchymal stem cells viability, directly inducing cellular differentiation promoting bone tissue regeneration by stimulating osteogenesis and angiogenesis [26]. These experimental data indicate that composites of CP and CS are promising materials for bone tissue engineering. It is critical to note that most of the cited studies employed synthetic procedures expected performing heat treatment of the obtained CP and CS composites, which transformed them into crystalline form. Biomimetic method of production a composite of β-CaSiO3 and poorly crystallized carbonated hydroxyapatite (CHA) by precipitation of apatite in the presence of solid wollastonite soaked in simulated body fluid were proposed in [27]. However, composites of amorphous salts such as poorly crystallized CHA and calcium silicate hydrates (CSH, rСаО·mSiO2·nH2O) that may exhibit increased biological activity as a result of enhanced chemical reactivity of disordered solid phases have not been fully investigated. In this study, an approach to synthesize composites of poorly crystallized CHA and CSH at different ratios by co-precipitation from aqueous solutions is attempted, and the behavior of composite ceramics in SBF is examined.

2.2. Bioceramics fabrication Powders of HA/CSH composite were used as starting materials to prepare bioceramics. 350 mg of the powder was compacted using a manual hydraulic tablet press “Carver 4350. L” under a pressure of 4 metric tons for 5–10 s in stainless-steel dyes. The resultant compacts were of 2 cm in diameter and 1 mm in height. 2.3. Powder characterization techniques All the resulting HA/CSH composite powders were examined using scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), laser diffraction, adsorption method (BET) and thermogravimetric analysis coupled with mass spectrometry (TGA/MS). Powder diffraction investigations (phase identification and scattering domains (CSD) measurements) were performed with a diffractometer “XRD-7000” (Shimadzu) at 40 kV and 30 mA using СuKα (λ = 1.54 Å) radiation as an X-ray source in the 2θ range from 10° to 60°, where almost all significant peaks of CP, CS and calcium carbonate (CC) appear. X-ray patterns were recorded with a 0.05° scan step and scanning speed of 4°/min. Identification of the crystalline phases was performed with the “SIeve+” software package using the PDF-4 database. CSDs were calculated with the Debye-Scherrer formula using the reflection at 25.8° for HA and 29.3° for CC. FTIR spectra were scanned on an IR spectrometer “FT-801” (Simex; Novosibirsk, Russia) using the KBr pellet technique in the range of 500–4000 cm−1 with 4 cm−1 and 32-fold scanning resolution. The program “ZaIR 3.5” was used to obtain and process the spectra. The results were interpreted according to the literature data [28–32]. Morphology and chemical composition of the samples was analyzed by scanning electron microscopy using a “JCM-5700” microscope (JEOL) equipped with an energy dispersive X-ray spectrometer “JED2300” in a high vacuum mode. The SpotSize parameter was selected as 10, 20 and 50. The accelerating voltage value ranged from 10 kV to 20 kV, and magnification ranged from 500× to 10,000×. HRTEM analysis of the obtained powders was performed on a “JEM2100” (JEOL) microscope with a resolution not < 0.134 nm at 1.5 × 108 fold magnification (200 kV). The microscope was equipped with an energy dispersive spectrometer “Inca-350” for local chemical microanalysis. It was also possible to obtain maps of the distribution of elements on a dedicated section of the TEM micrograph in the characteristic X-ray radiation (Kα). Sample's powder was dispersed into a colloidal solution in ethyl alcohol, sprayed onto a porous carbon film coated Cu-grid and placed into a microscope chamber after drying. Micrographs and maps of the distribution of elements from the selected part of TEM micrographs with characteristic X-ray radiation (Кα) were obtained for studied powders. The powder particle size distribution was determined by a laser diffraction using a particle size analyzer “SALD-2300” (Shimadzu). The complex refractive index for the measurements was 1.65 ± 0.00i. Differential curves for the particle size distribution were obtained for both average and modal particles and determined using the software package “WingSALD II” (Shimadzu). The texture characteristics of the powders were determined by the adsorption method using nitrogen adsorption-desorption isotherms at 77.4 K obtained on the analyzer “Gemini VII” (Micromeritics Instrument Corporation). The samples were outgassed in vacuum at 140 °C for 10–12 h before adsorption measurements. The specific surface area of the powder was determined using the BET method [33]. Thermal transformation processes of the composites were determined using the TGA/MS method on a thermal analyzer “STA-449C” (Netzsch) coupled with a mass spectrometer “Aeolos” (Netzsch). The samples were heated to 1000 °C at a rate of 10°/min in a stream of a mixture of 22 vol% air/argon (70 mL/min). Mass spectrometric

2. Materials and methods 2.1. Powder synthesis Composites with different HA/CSH weight ratios were synthesized using the method of chemical co-precipitation of poorly water-soluble salts from aqueous solutions. All reagents were of analytical grade and used for synthesis without further purification. Ca(OH)2, Н3РО4, and Na2SiO3·5H2O were selected as initial reagents to minimize formation of byproducts and impurities in precipitated solid phases. All syntheses were performed in plastic vessels. Concentration of the solutions of the initial reagents are shown in Table 1. The theoretical amount of HA in solids ranged from 0 to 100 wt%. The suspension of Ca(OH)2 with the corresponding concentration was stirred on a magnetic stirrer (“Big squid white”, IKA) for 1 min at 1250 rpm to disperse Ca(OH)2 uniformly. Solutions with the corresponding concentrations of Н3РО4 and Na2SiO3 were gradually (4–5 mL/min) added into the reaction mixture followed by addition of 20% NaOH solution to adjust pH to 12.0. The reaction mixture was allowed to stand for 22–24 h. Precipitated white solid was separated by filtration washed with three portions of distilled water (100 mL each), dried at 90 °C to constant weight and ground in a porcelain mortar to a powder form. Each experiment was carried out three times.

Table 1 Concentration (mol/L) of initial reagents used for synthesis of composites with different weight ratios of HA/CSH. Reagent concentration, mol/L

Н3РО4 Na2SiO3 Ca(OH)2

Theoretical composite ratio (HA/CSH, wt%) 100/0

80/20

60/40

50/50

40/60

20/80

0/100

0.060 – 0.100

0.048 0.017 0.097

0.036 0.034 0.094

0.030 0.043 0.093

0.024 0.052 0.092

0.012 0.069 0.089

– 0.086 0.086

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in aqueous solutions by ion exchange reactions as low-temperature precursor phases of wollastonite (WT, CaSiO3) [35]. Figs. 1 and 2 depict TEM micrographs and element distribution maps of a region of interest in characteristic X-ray radiation (Kα) for samples C8 and C2, showing that Ca, P, Si, C and O are determined in the powders. The results indicate that these elements are uniformly distributed over the material surface.

detection of inorganic products released under heating (CO, CO2, H2O, NO, NO2) was performed. 2.4. Chemical analysis of liquids Chemical analysis of liquid phases separated from precipitates was performed. The pH values were measured using a pH-meter “pH150MI” with a combined pH-electrode and thermal sensor. Residual Ca2+, PO43− and SiO32− concentrations in solution were determined by the photometric method on the spectrophotometer “UV-1200” (EcoView). Calcium was determined by the reaction with Arsenazo III in alkaline medium. The quantitative UV method for phosphate determination is based on the interaction between PO43− and ammonium molybdate in acidic medium to form molybdophosphoric heteropolyacid. Silicates were determined as yellow molybdosilicic acid, which forms by reaction of monomeric-dimeric forms of silicic acid and silicates with ammonium molybdate in acidic medium. Each determination was carried in triplicate. The composition of the precipitates was calculated as the difference between initial and final ions' concentrations in the mother liquor.

3.1.2. Phase composition In Fig. 3, the X-ray patterns of samples C10 - C0 are shown. According to XRD results, poorly crystallized HA and CC are identified in the powders. Apatite reflections in X-ray patterns are poorly resolved. Instead of the characteristic triplet at 32.2°, 34.0° and 35.4° 2θ, one unresolved peak appears in the 30–35° 2θ region. Its intensity decreases as the calculated apatite content in materials is decreased. Simultaneously, the reflections of calcite (CaCO3) become more evident (Fig. 3). The CS peaks were not detected on X-ray patterns of the composites. However, for sample C0, in addition to CC bands, a halo at 30°–35° 2θ was identified. According to [36], this feature is characteristic for CSHs with a closed amorphous structure.

2.5. Soaking in simulated body fluid (SBF)

3.1.3. FTIR analysis The IR spectra of powders are shown in Fig. 4. In spectra of C2 - C8 composites and sample C10, bands at 565, 605, 962, 1030 and 1060 cm−1 caused by O–P–O stretching vibrations correspond to HA [29]. Overlapping absorption bands in the region of 1030–1090 cm−1 indicate that poorly crystallized HA is formed under the experimental conditions which is consistent with XRD data (Fig. 3). Absorption bands of the bonds in siloxane bridges Si-O-Si (670 cm−1) and groups SieO (970 and 1060 cm−1) from the CSH structure appear in IR spectra of control sample C0 [30]. These modes are also detected in composites' spectra. Their intensity decreases simultaneously with the decrease of theoretical content of CS in the powders. Simultaneously, the strongest CSH reflection (ν = 970 cm−1) gradually merges with the absorption bands of phosphates (ν = 1030–1060 cm−1) and appear as a shoulder to them. For sample C8 with a minimal content of CS, silicon fragment bands in IR spectra are poorly distinguishable because they overlap with the phosphate band (ν = 962 cm−1). Vibrations of bonds SieO in amorphous SiO2 (ν = 800, 960, 1080, 1200 cm−1 [31]) that formed as a result of partial conversion of CSH to CC (Reaction (1)) do not appear in the IR spectra of the samples being of low intensity and/or overlapping with bands of calcium salts.

In order to study the ability to form apatite in vitro, tableted composite bioceramics were soaked in 50 mL of SBF as proposed by Kokubo [34]. SBF was prepared by dissolving analytical reagent grade NaCl (7.996 g), NaHCO3 (0.350 g), KCl (0.244 g), K2HPO4·3H2O (0.288 g), MgCl2·6H2O (0.305 g), CaCl2 (0.278 g) and Na2SO4 (0.071 g) in 1 L of distilled water with subsequent correction of pH to 7.4 with 1 N HCl. All experiments were performed at 37 °C under static conditions. Plastic containers with a flat bottom were used for all SBF preparations and soaking experiments. At certain time intervals (from 1 to 14 days), pH of the solutions as well as concentrations of Ca2+, PO43− and SiO32− ions were detected using chemical analysis methods (see Section 2.4). To maintain a constant volume, a portion of the fresh SBF was added to the system each time after an aliquot was removed for analysis. Each determination was carried in triplicate. After soaking, the tablets were removed from the SBF and dried at 37 °C. The formation of an apatite layer on the surfaces of the materials was evaluated. The microstructure and chemical composition of the samples were examined by SEM. 2.6. Statistical analysis

r CaO·mSiO2 ·nH2 O + r СО2 → r CaСO3 + mSiO2 + nH2 O

The experimental data was analyzed by Student's t-test method. All sample data was collected not less than in three replicates and expressed as mean ± standard deviation. A p value < 0.05 was considered as statistically significant.

(1)

3.1. Powder characterization

Along with the absorption bands of the groups that form the CP's and CS's crystal structure, spectra contain vibrations modes of CeO bond in CO32– ions (875, 1420, 1460 and 1540 cm−1), which are incorporated into CC's structure and partially replace the phosphate and hydroxyl positions in apatite producing AB-type carbonate substituted hydroxyapatite (CHA). CHA is obviously the main component of sample C10.

3.1.1. Chemical composition The chemical composition of synthesized powders is shown in Table 2. Experimentally determined values of Ca, P and Si content in the samples and the molar coefficients Ca/(P + Si) are close to the theoretical values corresponding to mixtures of HA and CS. Deviation of experimentally determined amounts of calcium, phosphorus and silicon in the obtained materials from the corresponding theoretical values might be attributed to the presence of byproducts. The closer the molar coefficients Ca / (P + Si) are to the theoretical values, the higher is the theoretical content of CS in the samples. In the control experiments (C10, C0), the powder stoichiometry corresponds to HA and CSH(I) (СSH0.8) phases. The latter is a member of the group of calcium silicate hydrates (CSH, rCaO·mSiO2·nH2O), which are formed

3.1.4. Particle size and morphology Powder particle size and specific surface area are presented in Table 3. According to the calculations based on the XRD data, CHA and CC crystallites have nanometer dimensions (CSH crystallite sizes were not calculated due to the absence of well-resolved XRD reflections for this phase). Such dimensions result in high values of the specific surface area for freshly precipitated samples. According to the laser diffraction (Table 3) and SEM (Fig. 5) data, the solid phases include particles of 3–200 μm in diameter and have median diameter from 60 to 100 μm. The significant difference in crystallite and powder particle sizes, as established by XRD and laser diffraction, indicates a high integration degree of the initial nuclei into mesoporous aggregates for CHA, CSH and CC. The CHA, CSH and CC

3. Results

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Table 2 Composition of the precipitates. Sample

WHA, wt%

W(Ca), wt%

C10 C8 C6 C5 C4 C2 C0

100 80 60 50 40 20 0

40.2 37.6 35.3 33.9 33.6 31.4 26.7

± ± ± ± ± ± ±

0.9 0.6 0.2 0.6 0.7 0.4 0.5

/ / / / / / /

39.8 38.6 37.3 36.7 36.1 34.9 33.6

W(Р), wt%

W(Si), wt%

Ca/(P + Si)

18.6 ± 0.3 / 18.5 14.5 ± 0.2 / 14.8 9.9 ± 0.1 / 11.1 8.5 ± 0.2 / 9.3 6.8 ± 0.1 / 7.4 3.3 ± 0.1 / 3.7 0

0 3.7 ± 0.2 / 4.7 8.8 ± 0.2 / 9.4 10.7 ± 0.7 / 11.8 12.7 ± 0.4 / 14.1 17.0 ± 0.3 / 18.8 18.8 ± 0.7 / 23.5

1.67 1.57 1.39 1.30 1.24 1.10 0.99

± ± ± ± ± ± ±

0.01a / 1.67 0.02 / 1.49 0.02 / 1.34 0.05 / 1.27 0.04 / 1.21 0.01 / 1.10 0.01b / 1.00

WHA – HA's ratio in the samples specified in the calculation, the remainder is CSH. The left number in the column is an experimental value, while the right one is a calculated value. a Ca/P value is indicated. b Ca/Si value is indicated.

Fig. 1. TEM micrographs and elemental distribution maps for a region of interest in characteristic X-ray radiation (Kα) for sample C8. Scale bar on micrograph of C8 is 500 nm.

region 650–750 °C corresponds to the dissociation process of CC [9]. The mass loss monotonously increases with the increase of the CS's ratio in the samples and changes from 1.0 to 6.3 wt% as a result of the CSH's dehydration process and from 0.8 to 5.3 wt% during decomposition of CC. DTG/MS curves show another carbonate peak that can be observed at t > 750 °C and attributed to the removal of carbonate ions as CO2 from CHA [29]. The mass loss in this temperature region ranges from 0.67 to 1.09 wt%, indicating a low degree of phosphate and hydroxyl ions substitution by carbonate in apatite. X-ray patterns of annealed samples C6 and C2 are shown in Fig. 7. Powders after heat treatment are two-phase composites and consist of Ca10(PO4)6(OH)2 and β-CaSiO3. Reflections of impurity phases are absent. This finding also confirms earlier conclusions about the composition of the freshly precipitated solid phases, particularly, the presence of apatite and CSH in the samples.

crystallites in these aggregates cannot be distinguished by SEM and HRTEM methods (Figs. 1, 2, and 5).

3.1.5. Thermal analysis Mass changes of freshly precipitated composites during calcination and composition of the gaseous products released under heating were studied using the TGA/MS method. Shown in Fig. 6 differential thermogravimetric curves were obtained for powders C10, C8, C2 and C0 heated in a stream of an air‑argon mixture. Samples' mass decreases in three temperature intervals (25–300 °C, 450–600 °C, and 650–800 °C). Mass spectrometric detection of gaseous inorganic products showed that dehydration of the solid phases and evaluation of CO2 molecules from their surface occur in a temperature range of 25–600 °C. The minimum value at t ≈ 100°С on differential thermogravimetric curves (DTG) corresponds to evaporation of adsorbed H2O molecules weakly bound to the surface. At this stage, the samples' mass loss is 8–12 wt%. The appearance of a peak's shoulder at t > 100 °C is caused by the removal of capillary condensed water in the aggregates' pores. The mass loss occurring at 450–600 °C is due to the release of crystallization water of CSH [37]. Molecules released at higher temperatures have molecular mass of 44 and identified as CO2. The minimum peak in the

3.2. Soaking in simulated body fluid (SBF) During the experiments of soaking ceramics in SBF, the solutions' composition was monitored. Fig. 8 shows Ca2+, PO43− and SiO32− ion concentrations and pH of the solutions depend on the soaking time of 4

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Fig. 2. TEM micrographs and elemental distribution maps for a region of interest in characteristic X-ray radiation (Kα) for sample C2. Scale bar on micrograph of C2 is 1 μm.

Fig. 3. X-ray patterns of composite powders.

composites' tablets and control samples in SBF. Changes in solutions' composition occur as a result of the interaction between the solid and liquid phases. For all studied materials, calcium and phosphate concentrations at each time point are lower than the initial content of these

Fig. 4. IR spectra of composite powders.

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Results of chemical composition analysis of the surface layer of tablets' upper side obtained by SEM mapping technique are presented in Table 4. According to experimental data, the studied materials contain Ca, P, Si, O and C as well as small amounts of Na+, Mg2+, and Cl− which could be captured from SBF and present in the samples as pore fluid. At the end of experiment, the calcium content on the tablet surface was lower than the initial value (Table 2), whereas, phosphorus content in samples C6 - C0 increased after soaking in SBF. The calculated values of Ca/P molar coefficients range from 1.2 to 2.2. These values are typical for amorphous CP [38]. Apparently, the decrease in silicon content in the samples to 2–4 wt% can be explained by initial dissolution of the CSH's phase followed by formation of the intermediary shielding layer of CP. SEM data shows that surface morphology of the lower sides of the tablets, which was in close contact with the reaction vessel's bottom in the process of experiments, significantly differs from the upper sides. Indeed, the surface of control samples C10 and C0 remains almost unchanged after soaking in SBF, and new morphological forms were not detected. In contrast, ceramics prepared from composite powders are coated with trigonal prismatic crystals growing on the surface base substrate. The density of such surface coating decreases as the theoretical content of CS increases in the sample. Particle size increases correspondingly. Crystals formed on C6 composite exhibit an average length of approximately 30 μm, and the length of trigonal particles on the C2 sample exceed 100 μm. To determine the composition of these crystals, local chemical analysis was performed. The main components of the particles are Ca (37.0 ± 2.5 wt%), C (12.1 ± 1.4 wt%) and O (50.3 ± 1.9 wt%). Consequently, CaCO3 crystals are formed on the lower side of the composite ceramics during the experiment. The chemical composition of tablets' lower side was determined by SEM mapping technique and is presented in Table 4. The surface layer of all studied ceramics contains Ca, P, O and C. Si was found in samples C6 - C0. A noticeable decrease in calcium content in the surface layer of all samples was detected after soaking in SBF compared with the initial value (Table 2) (a similar change was observed for the upper side of ceramics). Phosphorus and silicon levels on the surface of control samples C10 and C0, respectively, also decreased. Incidentally, phosphorus was found on the surface of pure CS ceramics, which can be attributed to the formation of some amount of CP during the experiment. Regarding composite samples, a simultaneous decrease was noted in P and Si levels compared with the initial values for these elements. However, as it goes from sample C6 to C2, content of these elements increases on the ceramic surface layer, while the content of carbon decreases. The described data correlate with the change in density of the material's surface coating induced by CC crystals and detected by SEM. The phase composition of the samples after soaking in SBF was

Table 3 The powder particle size and specific surface area. Sample

CSD(HА), nm

CSD(CC), nm

Dmedian, μm

SSA, m2/g

dpores, nm

C10 C8 C6 C5 C4 C2 C0

8.4 7.9 6.7 6.0 5.2 6.4 –

– – 3.2 4.6 4.1 8.2 6.9

94.3 68.9 63.5 80.4 88.1 99.3 62.2

90 67 101 100 117 123 101

– 8.6 10.5 10.7 8.4 – 8.6

ions in SBF (see [34]). Throughout the experiment, concentration of Ca2+ and PO43− ions decrease in the medium. The higher the current calcium concentration, the smaller theoretical content of HA in ceramics. The residual phosphate levels in solutions in contact with composites do not significantly differ. After 4 days of experiments, the systems contain approximately 48 μmol/L of PO43−. It was found that exposition of the reference samples to SBF provides higher PO43− ions concentrations in solutions: approximately 0.1 mmol/L for C10 and 0.23 mmol/L for C0. Whereas, for composites and samples C0, SiO32− ion concentrations and pH of solutions at the initial stage of experiments (within the first two days) rapidly increase (Fig. 8c and d). Over time, the rates of change of these parameters decrease. As the CS's theoretical ratio increases in composites, the current values of silicate ion concentrations change nonmonotonically. The smallest deviation in solution pH from the initial pH of SBF was detected for sample C10. In experiments with composites and sample C0, the pH increases to 8.5–8.9. After 14 days of soaking in SBF, the morphology of the upper side of the tablet that interacts with the bulk of solution and the lower side that serves as the vessel bottom were examined. Micrographs of the tablet surface before and after interaction with SBF are shown in Fig. 9. Surface morphology of the upper and lower sides of the ceramics is shown in Figs. 9b, c. Development of the spherical particles on the upper side of the ceramics was observed during the experiment (Fig. 9b). Their quantity and size increase as the content of CS in materials increases. Thus, single granules are observed on the C10 sample, while a significant part of the C0 tablets' surface is covered with bulk agglomerates of spherical particles. The structure of the base substrate to which particles are attached differs from the initial structure of ceramics. The boundaries between grains of CS are not visible, and the surface is smooth. Local analysis of the granules' chemical composition showed that the main components include Ca (32.3 ± 4.1 wt%), P (21.9 ± 2.3 wt %) and O (36.5 ± 5.6 wt%). The particles also contain 4.4 ± 1.2 wt% carbon, suggesting that a layer of spherical particles of amorphous CP with isomorphically included CO32– ions is formed on the ceramics' surface during soaking in SBF.

Fig. 5. Micrographs of samples C10 (a) and C2 (b). 6

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Fig. 6. Differential thermogravimetric curves of samples C10, C8, C2 and C0.

ions was formed on the ceramic surface during the experiment. Traces of silicate components and CC from the composition of initial tablet's material were detected by peaks at 800 and 712 cm−1, respectively. Calcite forms on the other side of composite ceramics. A set of intense characteristic calcite peaks with maxima at 712, 875, 1420, and 1800 cm−1 [32] are noted in FTIR spectra. 4. Discussion Aqueous solutions, concurrently containing Ca2+, PO43−, SiO32−, OH−/H+ ions, are complex systems where cocrystallization of several poorly soluble compounds is possible under certain conditions [39]. The conditions for precipitation of the salts can be selected considering ionic equilibriums in solutions containing phosphate and silicate ions (Fig. 12). For example, Na2SiO3, which was used in this work as an initial reagent in synthesis, is a salt of weak dibasic metasilicic acid. Dissociation of it leads to formation of SiO32−, HSiO3−, and H2SiO3 in the aqueous medium. The ratio of these silicate forms strongly pH dependent. Fig. 12 demonstrates that at pH ≤ 8 silicates exist in a lowactive molecular form (H2SiO3) which transforms into silica gel (nSiO2·mН2О) and precipitates upon aging. As the pH increases, the ratio of the silicate anionic forms also increases (pH = 9: 18% HSiO3− and 82% H2SiO3; pH = 10: 68% HSiO3− and 31% H2SiO3). The simultaneous presence of Ca2+ and HSiO3− ions in the system makes it possible the precipitation of CSH's phase along with amorphous SiO2. In a highly alkaline medium (pH ≥ 11), metasilicic acid is present in the systems in trace amounts; > 95% of all silicates exist as SiO32− and HSiO3−. The reaction of these ions with calcium ions leads to the formation of CSH's phase in alkaline solutions [39,40]. Phosphoric acid used as an initial reagent in this work is also polybasic. It is capable to stepwise dissociation with the formation of Н2РО4−, НРО42− and РО43−. The ratio of these ions in solutions (Fig. 12) and the stoichiometric composition of the synthetic CPs depend on pH: Са(Н2РО4)2·2Н2О (рН = 0–2), СаНРО4·2Н2О (рН = 2–6), Са8(НРО4)2(РО4)4·5Н2О (рН = 5.5–7), Са10-х(HPO4)x(РО4)6-x(ОН)2-x or Са10(РО4)6(ОН)2 (рН = 6.5–12) [1]. Consequently, synthesis of CSH and apatite phases should be carried out in alkaline media. Besides, described properties allows polycomponent materials to be obtained. In this study, a series of samples was synthesized in alkaline solutions with different concentrations of calcium, phosphates and silicates. The resulting freshly precipitated solids were investigated by a group of physicochemical methods without prior annealing. It was established that the solid phases contain various amounts of Ca, P, Si, O and C,

Fig. 7. X-ray patterns of annealed samples C6 and C2.

tested by XRD. X-ray patterns of the upper side of C2 composite tablets with the highest quantity of spherical CP particles and the lower side of C6 composite ceramics, which was almost completely covered with prismatic CC crystals, are shown in Fig. 10. X-ray patterns of composite materials before and after contact with SBF contain CC peaks (in calcite form) and poorly crystallized CP. An increase in CP reflection intensity with respect to CC peaks was observed by comparing X-ray patterns obtained for initial ceramics and their upper side after soaking in SBF (Fig. 10a). Based on SEM data, this finding can be attributed to the CP's layer formation on the tablet surface during experiments. Opposite changes were derived from the analysis of the X-ray patterns from the lower side of composite ceramics (Fig. 10b). For the samples exposed to SBF for 14 days, calcite peak intensity significantly increases because the tablet's surface is almost completely covered by CaCO3. FTIR spectra of surface layer formed on the upper and lower sides of composite tablet C4 during 14-day soaking in SBF are shown in Fig. 11. The composition of these two layers varies significantly. The spectra of a substance taken from the upper side of ceramics contains peaks characteristic for HA (565, 605, 962 (shoulder), 1040 cm−1). Unresolved bands in the region from 1030 to 1090 cm−1 and the presence of the intense carbonate absorption bands (875, 1420, and 1450 cm−1) indicate that poorly crystallized CP with isomorphically included CO32– 7

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Fig. 8. Dependence of (a) Ca2+, (b) PO43− and (c) SiO32− ion concentration, and (d) pH on soaking time in SBF of the tablets prepared from HA/CSH composites.

С0, a halo at 30°–35° 2θ was observed. The presence of halo is characteristic for CSH [36,40]. Overlapping of the apatite and composite Xray patterns does not allow the definite determination of rCaO·mSiO2·nH2O in freshly precipitated powders by XRD. However, the presence of CSH may be indicated by CC impurity detected by XRD and FTIR. So far, simultaneous formation of Ca10(PO4)6(OH)2 and CaCO3 in solutions without the addition of CO3-containing reagent has not been reported in the literature. However, CC is formed as an impurity during CSH precipitation in open alkaline systems [30]. At these conditions, carbonic acid is formed as a result of CO2 absorption from air by a solution. Since carbonic acid is a stronger acid than metasilicic acid, the former is able to displace the latter from its salts. Thus, due to anion exchange, a partial conversion of precipitated CSH to CC occurs (reaction (1)). The more intense CaCO3 reflections in X-ray patterns (and, accordingly, the higher its content in precipitates), the higher the ratio of CS in powders (Fig. 3). Consequently, there is a positive correlation between the amount of calcium silicate and calcium carbonate that is formed. The described results of the synthetic samples' composition indicate the formation of polyphase materials at the experimental conditions. Powders consist of poorly crystallized CHA and CSH with CC as an admixture. Additional HRTEM analysis showed that salts' components (Ca, P, Si, C and O) are uniformly distributed over the material surface (Figs. 1, 2). This fact indicates that the salts form homogeneous composites in the studied systems during the crystallization process. During deposition process, CHA, CSH and CC nanocrystallites are combined into bulk mesoporous aggregates, supported by the fact that synthetic powders have high specific surface area values (Table 3). TGA/MS investigations revealed that the processes of H2O and CO2 molecules release from composite samples during annealing lead to the formation of crystalline phases Ca10(PO4)6(OH)2 and β-CaSiO3 (Fig. 7). The only phase found in the composition of reference sample C10 is HA, whereas powder C0 exclusively contains WT. Composite samples C2 -

depending on the initial concentrations of precipitating ions in the reaction medium. The calculated values of Ca/P and Ca/Si molar coefficients for control samples C10 and C0, respectively, and Ca/(P + Si) for composites were close to the theoretical values characteristic for HA and CSH(I) mixtures. The presence of CP in the freshly precipitated solids was reliably determined by XRD and FTIR. Since their reflections in X-ray patterns and absorption bands of O–P–O bonds in IR spectra are poorly resolved, it indicates that poorly crystallized apatite was formed at experimental conditions. This finding is consistent with [41], where HA with a low degree of crystallinity formed as a result of precipitation in aqueous solutions at room temperature even at high pH levels and a stoichiometric ratio of Ca- and P-containing reagents used. In the IR spectra of samples containing CP (C10 - C2), the modes of CeO bond vibrations maxima present at 875, 1430 and 1470 cm−1 which are characteristic absorption bands for CO32– ions partially replacing the PO43− groups in apatite. Thus, poorly crystallized carbonate-hydroxyapatite (CHA) is precipitated at the experimental conditions. Literature data states [42] that the process in which phosphates and hydroxyl ions are replaced by carbonate ions can be described as follows:

0.5 Ca2 + + РО43 − ← 0. 5□Ca + CО32 −

(2)

2 ОН− ← CО32 −

(3)

Here, we can notice that amounts of Ca and P in the composition of the solid phase decrease simultaneously in case of B-type substitution. Considering the low degree of substitution of PO43− ions by СО32− (< 1.1 wt%, according to TGA/MS data), this may lead to the formation of apatite with molar coefficient Ca/P close to 1.67 at the conditions С10 - С2 samples were synthesized. The silicate component in freshly precipitated samples is detected by the absorption bands of SieO bonds from the CSH structure in the IR spectra. Due to its amorphous structure, well-resolved reflections of these phases were not detected on X-ray patterns. However, for sample 8

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Fig. 9. Micrographs of the surface of the ceramics prepared from HA/CSH composites after 14-day soaking in SBF.

powders' initial components can be described by reactions (4)–(7):

C8, which contained varying amounts of CHA and CSH with CC admixture before calcination, exclusively consist of Ca10(PO4)6(OH)2 and β-CaSiO3 in theoretical proportions after heat treatment (20–80 wt% of apatite and remaining WT, Table 2). Such transformation of the 9

Ca10 − х/2 (РО4 )6 − x (CО3 ) x + y (ОН)2 − 2y → Са10 (РО4 )6 (ОН)2 + хСO2 ↑

(4)

rСаО·mSiO2 ·nH2 O → β − CaSiO3 + nH2 O↑

(5)

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Table 4 Composition of the surface layer formed on the upper and the lower sides of the composite tablets after 14-day soaking in SBF. W, wt%

Sample С10

С6

С5

С4

С2

С0

Upper side Ca P Са/Р Si O C Na Mg Cl

22.2 ± 1.0 14.1 ± 0.3 1.22 – 51.1 ± 1.1 10.8 ± 0.2 0.9 ± 0.1 0.4 ± 0.1 0.4 ± 0.1

19.0 ± 0.9 10.7 ± 1.3 1.38 2.6 ± 0.7 53.9 ± 1.7 12.2 ± 0.5 0.7 ± 0.1 0.7 ± 0.1 0.4 ± 0.1

16.2 ± 0.7 9.6 ± 0.3 1.31 1.5 ± 0.2 55.3 ± 0.7 15.5 ± 0.4 0.8 ± 0.1 0.7 ± 0.1 0.4 ± 0.1

26.5 ± 0.4 11.7 ± 0.4 1.67 2.4 ± 0.5 46.2 ± 0.2 10.9 ± 0.5 0.9 ± 0.1 0.7 ± 0.1 0.7 ± 0.1

27.6 ± 1.3 9.8 ± 0.6 2.17 3.7 ± 0.2 44.9 ± 0.4 11.2 ± 0.2 1.0 ± 0.1 0.5 ± 0.2 0.9 ± 0.2

25.8 ± 1.2 13.5 ± 1.0 1.49 2.4 ± 0.4 45.5 ± 0.9 4.1 ± 0.7 3.5 ± 0.2 1.8 ± 0.2 3.4 ± 0.5

Lower side Ca P Si O C Na Mg Cl

28.2 ± 1.4 16.1 ± 1.3 – 45.8 ± 1.4 8.2 ± 1.5 0.9 ± 0.2 0.5 ± 0.2 0.4 ± 0.1

27.5 ± 0.7 0.5 ± 0.1 0.2 ± 0.1 53.9 ± 1.3 17.9 ± 1.7 – – –

27.1 ± 0.4 0.8 ± 0.1 0.5 ± 0.1 56.1 ± 2.3 14.8 ± 0.4 – 0.7 ± 0.2 –

28.9 ± 1.1 1.6 ± 0.5 2.2 ± 0.3 54.2 ± 2.6 13.0 ± 1.2 – – –

25.1 ± 0.8 2.2 ± 0.4 9.3 ± 0.8 51.7 ± 1.5 10.7 ± 0.9 0.5 ± 0.2 0.5 ± 0.1 –

17.0 ± 0.3 1.1 ± 0.2 15.2 ± 1.2 54.7 ± 0.9 10.3 ± 1.4 0.8 ± 0.1 0.6 ± 0.2 0.4 ± 0.1

Fig. 10. X-ray patterns of the upper side of the composite tablet С2 (a) and of the lower side of composite tablet С6 (b) before and after 14-day soaking in SBF. * calcite.

tricalcium phosphates (TCP, β-Ca3(PO4)2, α-Ca3(PO4)2) mixed with calcium pyrophosphates (CPP, β-Ca2P2O7, α-Ca2P2O7) or with stoichiometric HA. As a single phase, the latter is formed at temperatures below 1300 °C from precipitated apatite with a molar coefficient of 1.67. Apatite with higher Ca/P ratios decomposes into Ca10(PO4)6(OH)2 and CaO upon calcination. The absence of TCP,CPP and/or CaO peaks on X-ray patterns of calcined composites indicates that apatite obtained under experimental conditions has a molar ratio Ca/P close to 1.67 because its thermal conversion proceeds according to reaction (4). The composition of CSH calcination products also depends on salts' stoichiometry [35]. WT is formed after annealing of CSH with Ca/ Si = 1 at t > 800° С. Samples with Ca/Si < 1 and Ca/Si > 1 transform after heating into a mixture of SiO2 or Ca2SiO4 with βCaSiO3, respectively. Consequently, the fact that only crystalline silicon-containing phase (WT) was found by XRD in the calcined powders indicates that CSH with Ca/Si ≈ 1 precipitates along with apatite at the experimental conditions. CaCO3 is formed during synthesis (reaction (1)) as an admixture to calcium phosphate and calcium silicate. CC is decomposed by reaction (6) under heating at temperatures of approximately 700 °C [9]. In this regard, CaO appearance in the calcination products of freshly precipitated samples was expected. However, calcium oxide reflections were not observed in X-ray patterns of calcined materials (Fig. 7). As a result of reaction (1), some amount of SiO2 appears together with CaCO3 in powders, but SiO2 peaks were also not detected in X-ray

Fig. 11. FTIR spectra of the surface layer formed on the upper (a) and the lower (b) sides of composite tablet C4 during 14-day soaking in SBF.

CaСO3 → СаО + СO2 ↑

(6)

СаО + SiO2 → β − CaSiO3 (or CaСO3 + SiO2 → β − CaSiO3 + СO2 ↑)

(7)

The composition of apatite's calcination products depends on the Ca/P value, as indicated in Table 5. Heat treatment of samples with Ca/ P < 1.67 at temperatures higher than 700 °C leads to the formation of 10

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Fig. 12. Ion diagrams of metasilicic and phosphoric acids. ○ - H2SiO3, ● - HSiO3−, ● - SiO32−, Δ - H3PO4, ▲ - H2PO4−, ▲ - HPO42−, Δ - PO43−.

patterns of calcined materials. This finding may be due to the crystallization of β-CaSiO3 (reaction (7)). Thus, the data of chemical analysis and physicochemical investigation of freshly precipitated and calcined powders is consistent and indicates the formation of composites of poorly crystallized CHA and CSH with an admixture of CC in experimental conditions. Possible perspectives in application of the obtained compositions to bone restoration in medicine are inferred on their ability to dissolve gradually and initiate processes of bioapatite formation is of great importance. A method based on materials soaking in a prototype of human blood plasma (SBF) is often used for preliminarily evaluation of sample properties at the laboratory conditions. This experiment was performed for the ceramic samples from synthetic polyphase powders. The results of this experiment showed that the chemical composition of solutions varies with tablet contact time due to the processes of resorption and secondary precipitation (Fig. 8). According to CP and CS dissolution mechanisms [48,49], the exchange of Ca2+ ions from solid phases to protons from solution occurs as salts interact with an aqueous medium, leading to alkalinization of all studied systems (Fig. 8d). A significant change in pH (relative to the initial acidity of SBF) as well as SiO32− ion appearance in media in contact with silicon-containing samples (Fig. 8c) indicates active dissolution of CSH in materials. The increase in current calcium concentrations in systems based on increased CS proportions in ceramics (Fig. 8a) is obviously due to the higher solubility of the CSH phase compared with HA. The ongoing processes of ceramic component transition to the solution however do not lead to the accumulation of Ca2+ and PO43− in the medium. In contrast, calcium and phosphate concentrations at each period of time are less than their initial levels in SBF for all materials. The logical explanation for this experimental observation is that ions are consumed in the CP formation process. Formation of the CP's layer on the upper side of the ceramics was confirmed by SEM, XRD and FTIR (Figs. 9b, 10a, 11, and 12). Moreover,

the denser and the thicker the salt's layer was, the higher CSH content in was observed in ceramics. Individual spherical particles of a new phase were found on C10 and C6 tablets' surfaces. In addition, C0 samples' surfaces exhibited formation of the massive coating with deep cracks as a result of the CP's grains layering and growth (Fig. 13). Such surface variation of ceramics with different compositions is caused by the fact that a hydrated silica gel layer is formed during silicate phase dissolution as Ca2+ ions from the salt are exchanged with protons in the solution. Si-OH groups in this layer act as catalysts for CP crystallization [50]. Thus, the higher the CS content in material, the greater the number of active sites (Si-OH) formed. In addition, a more active ceramic remineralization process occurs due to the constant replenishment of Ca2+ ions leaving the CSH during resorption. In addition to the formation of CP, CaCO3 and CaSO4 theoretically can form in SBF. Calculations of solution supersaturation and the Gibbs free energy changes of apatite, CC and calcium sulfate during their crystallization in SBF showed that experimental solutions with immersed ceramics were undersaturated with respect to calcium sulfate throughout the experiment, while CC crystallization is thermodynamically possible (Table 6). Crystals of this phase were found on the lower side of tablets, which was exposed to the vessel's bottoms during all the experiments conducted (Fig. 9). Moreover, the smaller CC particles on the materials' surface were formed, the higher was the CSH's content. Chemical composition analysis of the lower side of tablets also indicated the presence of phosphorus on CSs' ceramics likely in the form of CP. On this basis, changes taking place on the tablet's surfaces can be schematically described. A layer of hydrated silica gel forms on both surfaces of a tablet as a result of CSH hydration, when samples C6 - C0 are immersed in solution. Then, poorly crystallized CP with isomorphically included CO32– groups is formed. On the upper side of ceramics, this process proceeds more effectively as CSH levels in the sample increase. As a result, the phosphate concentration in solution decreases. It can also be assumed that the higher CS content in the solid

Table 5 The composition of the samples obtained by calcination of apatite with different stoichiometry. Molar ratio Са/Р

Calcined samples composition

Calcination temperature

1.40 < Са/Р < 1.50 Са/Р = 1.50 1.50 < Ca/P < 1.67

Ca2P2O7 + β-Ca3(PO4)2 β-Ca3(PO4)2 β-Ca3(PO4)2 + Са10(РО4)6(ОН)2 α-, β-Ca3(PO4)2 + Са10(РО4)6(ОН)2 Са10(РО4)6(ОН)2 Са10(РО4)6(ОН)2 + CaO

t t t t t t

Ca/P = 1.67 Ca/P > 1.67

11

≈ 700 °C > 700 °C > 700 °C > 1200 °C < 1300 °C > 1000 °C

Ref. [43] [1] [43–46] [44,45] [43,46] [43,46,47]

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Fig. 13. a. Micrographs of the surface of ceramic C0 after 14 days of soaking in SBF. * b - magnification ×1000; markers indicate the points at which the composition analysis was performed (26.2 ± 3.4 wt% Са, 26.6 ± 3.9 wt% Р, 60.0 ± 5.7 wt% О, 4.73 ± 1.8 wt% С).

calcium phosphate layer formation occurs on the surface of control samples, containing the apatite or CSH. In the latter case, the most massive coating with deep cracks forms on the tablet surface. For composite samples, the formation of agglomerates of CP's spherical particles on the ceramics upper side and prismatic calcite crystals on the surface of the lower side, which was at small-volume confinement conditions during all the experiments, were discovered.

Table 6 Solutions supersaturation and the Gibbs free energy change of apatite, calcium carbonate and calcium sulfate during their crystallization in SBF after 7 days of synthetic ceramics soaking. Parameter

Sample C10

C6

С5

C4

C2

C0

Са10(РО4)6(ОН)2 Sa,b,c 20.1 ΔG, kJ/mol −6.37

12.6 −3.99

20.6 −6.53

19.3 −6.12

23.7 −7.52

23.5 −7.45

26.9 −8.51

СаСО3 S ΔG, kJ/mol

−0.35 1.01

−0.47 1.35

0.69 −1.98

0.58 −1.65

0.96 −2.75

0.94 −2.69

0.87 −2.47

CaSO4 S ΔG, kJ/mol

−2.07 5.90

−2.78 7.94

−2.32 6.61

−2.38 6.80

−2.26 6.45

−2.22 6.34

−2.17 6.20

SBF

Data availability statement The raw/processed data required to reproduce these findings cannot be shared at this time due to time limitations. Declaration of competing interest The authors declare that they have no conflict of interest.

a

S - solution supersaturation with respect to low soluble compound, calculated according to [51]. b ΔG - the Gibbs free energy changing in low soluble compound crystallization process, calculated according to [51]. c Supersaturation calculated for SBF composition, proposed by Kokubo [34], before ceramics' immersion.

References [1] S.V. Dorozhkin, Multiphasic calcium orthophosphate (CaPO4) bioceramics and their biomedical applications, Ceram. Int. 42 (2016) 6529–6554, https://doi.org/10. 1016/j.ceramint.2016.01.062. [2] G.R. Owen, M. Dard, H. Larjava, Hydoxyapatite/beta-tricalcium phosphate biphasic ceramics as regenerative material for the repair of complex bone defects, J. Biomed. Mater. Res. B. 106 (2018) 2493–2512, https://doi.org/10.1002/jbm.b.34049. [3] A.P. Solonenko, O.A. Golovanova, Hydroxyapatite - brushite mixtures: synthesis and physicochemical characterization, Rus. J. Inor. Chem. 59 (2014) 9–16, https:// doi.org/10.1134/s0036023614010173. [4] A.H. Touny, M.M. Saleh, Fabrication of biphasic calcium phosphates nanowhiskers by reflux approach, Ceram. Int. 44 (2018) 16543–16547, https://doi.org/10.1016/ j.ceramint.2018.06.075. [5] Z. Mohammadi, M.M.A. Sheykh, F. Rasouli, M. Noori, Biphasic calcium phosphates of hydroxyapatite/octacalcium phosphate with mixed morphology for hard tissue engineering application, J. Appl. Chem. 9 (2015) 31–40. [6] T.V. Safronova, V.I. Putlyaev, Y. Filippov, S.A. Vladimirova, D.M. Zuev, G.S. Cherkasova, Synthesis of calcium-phosphate powder from calcium formiate and ammonium hydrophosphate for obtaining biocompatible resorbable biphase ceramic materials, Glas. Ceram. 74 (2017) 1–6, https://doi.org/10.1007/s10717017-9958-4. [7] H. Okano, Y. Inomata, S. Nakagawa, Preparation and characterization of biphasic calcium phosphate consisting of tetracalcium phosphate and α-TCP, Phosphorus Res. Bull. 32 (2016) 14–16, https://doi.org/10.3363/prb.32.14. [8] L. Xie, H. Yu, Y. Deng, W. Yang, L. Liao, Q. Long, Preparation, characterization and in vitro dissolution behavior of porous biphasic α/β-tricalcium phosphate bioceramics, Mater. Sci. Eng., C. 59 (2016) 1007–1015, https://doi.org/10.1016/j. msec.2015.11.040. [9] M.A. Goldberg, V.V. Smirnov, V.M. Ievlev, S.M. Barinov, S.V. Kutsev, T.V. Shibaeva, L.V. Shvorneva, Influence of ripening time on the properties of hydroxyapatite calcium carbonate powders, Inorg. Mater. 48 (2012) 181–186, https://doi.org/10. 1134/S0020168512020094. [10] C.J.S. Ibsen, D. Chernyshov, H. Birkedal, Apatite formation from amorphous calcium phosphate and mixed amorphous calcium phosphate/amorphous calcium carbonate, Chem. Eur. J. 22 (2016) 12347–12357, https://doi.org/10.1002/chem. 201601280. [11] C. Liang, Z. Li, D. Yang, Y. Li, Z. Yang, W.W. Lu, Synthesis of calcium phosphate/

phase allows fewer CO32– ions in the solution interact with ceramic given their participation in the process of CHA formation. Besides, the lower side of tablets was in contact with a limited SBF volume. The concentration of phosphate ions in this part of system rapidly decreased, while carbonates remain in excess (С(СО32−) / С(РO43−)initial = 4.2). It potentially leads to an increased local supersaturation with respect to CC and further to its crystallization on the lower side of ceramics. The expected decrease of carbonate concentration in systems with C6 - C0 ceramics causes a consequent reduction of the solutions' supersaturation with respect to CC and, as a result, the decrease of CaCO3 crystals' quantity and the increase in their size.

5. Conclusion In this study, a series of powders containing from 0 to 100 wt% of poorly crystallized carbonate substituted hydroxyapatite (Ca/P ratio close to 1.67) mixed with calcium silicate hydrate (Ca/Si ≈ 1) and calcium carbonate admixture were obtained by salts' co-precipitation in aqueous solution. Solid phase particles are mesoporous aggregates of nanocrystalline calcium salts. Synthetic samples were characterized by high specific surface area values (from 67 to 123 m2/g). The results of in vitro experiments on ceramics soaked in SBF indicate that amorphous 12

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[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

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