Properties of calcium-phosphate materials obtained by sol-gel method: effect of MgO concentration

Abstract A calcium-phosphate system was obtained by sol-gel method from 0.4 M solutions based on ethyl alcohol, tetraethoxysilane, phosphoric acid, calcium nitrate, and magnesium nitrate. Two compositions with different content of CaO and MgO were prepared. After the maturation of solutions they followed by heat treatment at 60°C for 30 minutes and at 600°C and 800°C for 1 h. Solution with 20 wt.% MgO is suitable for film production up to seven days, while 5 wt.% MgO is available for creation of films only on the second day of its preparation. Thin films were obtained on the second day by spin-coating with a centrifuge speed of 3000 rev/min, followed by heat treatment at 60°C for 20 minutes and at 800°C for 1 h. The formation of crystalline phases in synthesized materials occurs at 800°C. An increase in the content of magnesium oxide in the system helps to accelerate the growth of particles on the surface of the samples when immersed in a simulation body fluid.


Introduction
Biomaterial is non-viable material intended for interaction with living tissue and performing medical functions. Three primary classes of biomaterials are mainly used as implants: metals, ceramics, and polymers. Calcium phosphate materials are of the greatest practical interest due to their high biocompatibility [1][2][3][4]. Due to good bioactivity, osteoconductivity, and biodegradability, calciumphosphate materials are used as bone repair materials of fillers since they form a bond to living bone for more than ten years.
The most suitable method for calcium-phosphate materials preparation is sol-gel synthesis since it allows obtaining materials with different properties: fine crystalline and coarse-grained materials by changing initial reagents technological conditions [5][6][7].
Glass ceramics based on the SiO 2 -P 2 O 5 -CaO system possess high bioactivity [8] depending on magnesium oxide additives. The relevance of magnesium for biomedical research is related to its fundamental role in cellular processes and human metabolism [9]. It is known that Mg stimulates the proliferation of osteoblasts and the ability of bone mineralization. Several studies have shown that divalent cations (Mg 2+ ) play a key role in bone remodeling and skeletal development [10,11]. Deficiency of Mg can be associated with a decrease in osteoclastic and osteoblastic activity, which leads to bone fragility and bone growth.

Experimental part
To obtain thin-film and dispersed materials based on the SiO 2 -P 2 O 5 -CaO-MgO system, a film-forming solution was prepared. Ethyl alcohol of 96 wt.% was used as a solvent, and calcium nitrate and magnesium nitrate were added with occasional stirring until complete dissolution of the salts. Then phosphoric acid and tetraethoxysilane were added to the solution, with a total concentration of each substance 0.4 M.
For the preparation of solutions based on the SiO 2 -P 2 O 5 -CaO-MgO system 2 compositions with different content of CaO and MgO were chosen (table 1). After ripening of the film-forming solution, thin-film and dispersed materials were obtained. Thinfilm materials were obtained by centrifuging on an MPW-340 centrifuge at a speed of 3000 rpm on silicon substrates (as a reference), then a step heat treatment was carried out until the formation of a thin film. Drying was carried out at a 60°C for 40 minutes followed by linear heating to 800°C. The samples were kept at 800°C for 1 hour prior to gradual cooling in the conditions of natural cooling of a muffle furnace. Dispersed materials obtained by drying the solutions at 60°C, followed by linear heating to 800°C.
The kinematic viscosity of the solutions was determined in a HPLC-2 capillary viscometer. The thermo-gravimetric analysis was performed on a NETSCH Jupiter STA 449 F1 thermal analyzer in the temperature range from 25 to 1000°C with a heating rate 30°/min in air atmosphere. The control of the evolved gas phase decomposition products was carried out with a NETZSCH QMS 403 D Aëolos mass spectrometer. IR spectra of dried solutions were reordered on a Agilent Cary 630 FTIR IR spectrometer in the frequency range of 400…4000 cm -1 . Phase composition of the dispersed materials was determined by X-ray diffraction on Rigaku MiniFlex 600 diffractometer (CuK α radiation). Surface morphology of the obtained materials was studied using a scanning electron microscope (SEM) HITACHI TM-3000. Elemental composition was determined by X-ray microanalysis by using a Quantax-70 instrument. The thickness of the films was measured on a laser ellipsometer Sentech SE400adv. The measurements were carried out at three points along the entire surface of the film. Evaluation of the bioactivity of the obtained materials was studied in vitro by keeping the samples in a cell-free simulation of simulation body fluid (SBF) for 14 days. Samples were placed in SBF at a 37 ± 0.5°C and pH = 7.4. The solution was changed every day for 14 days. The composition of the SBF solution is described in [12].

Results and discussion
Investigation of physicochemical processes occurring in solutions after their preparation was carried out by measuring the viscosity. On the first day, the viscosity of solutions has a value in the range from 1.77 to 1.82 mm 2 /s depending on solution' composition (table 2). On the second day, a significant decrease in viscosity value was observed in solution Mg5, and on the third day a white finely dispersed precipitation was precipitated. The Mg20 solution is stable for 13 days. At 14 th day, a precipitate forms in the Mg20 solution. Thus, with increasing magnesium content in the system, the stability of solutions increases.
Precipitation in 5 wt.% MgO solution is caused by hydrolysis and polycondensation processes [13,14] and electrostatic interaction between dissolved ions and solvent molecules, which can lead to both an increase and a decrease in the viscosity value. Since the viscosity of film-forming solutions 3 decreases in time, and hydrolysis and complexation reactions are characterized by high rate constants (i.e., they proceed almost instantly), the formation of the above solution can occur only due to the forces of electrostatic interaction of solutes between themselves and solvent molecules -ethyl alcohol. The decrease in the viscosity value of film-forming solutions in the first few days may be due to a weakening in the electrostatic interaction between solutes. Then the viscosity increases, since polycondensation processes begin to predominate in the solution. Regardless of the solution composition ( figure 1 a, 1b) (table 3).  [7,12], is formed as a result of hydrolysis and polycondensation of tetraethoxysilane.
The thermal analysis of dried solutions and the X-ray phase analysis of the products of their thermal destruction have allowed us to establish the formation processes of the samples as optimal temperature of synthesis (figure 2) and the composition of substances formed during the thermolysis of dried solutions (figures 5 and 6). The process of the material forming occurs in three stages ( figure  2). The first stage is at the temperature from 50°C to 200°C. It is corresponding to an endothermic effect in the DSC curve at 104°C. It is due to the removal of physically and chemically bounded water and it is accompanied by a large mass loss. The second stage (with less weight loss) is commenced after the end of the first stage (at 200°C) and run on until 600°C. It is corresponding to an exothermic peak at 230°C. It is probably related with the loss of nitrates and organics (i.e. alkoxy group). The     According to ellipsometry data, increase of MgO content in the system leads to the decrease of films' thickness (table 4). To study the bioactive properties, the samples were immersed in a solution of SBF for 14 days.  The study of the morphology of the coatings obtained after immersion in the SBF solution using electron scanning microscopy revealed large loose particles on the surface. Spherical particles with a size of 1.9-3.6 nm are fixed on the surface after holding the samples for 3 days (figure 7b, 8b). With an increase a storage time up to 7 days, the growth of needle particles in the surface is fixed (figure 7c, 7c). At 14 days holding samples in solution, the particles continue to grow and increase in size and reach 17.8 -30 nm ( figure 7d, 8d). The presence of needle particles contributes to the formation of a network structure on the surface of the samples, which is favorable for the further in growth of bone tissue in them and the formation of a stronger implant-bone connection. Increasing the magnesium content in the system helps to accelerate the growth of particles on the surface of the samples.

Conclusion
Glass ceramics based on SiO 2 -P 2 O 5 -CaO system possess high bioactivity [8] depending on the addition of magnesium oxide. Thus we synthesized dispersed materials and thin films based on the system SiO 2 -P 2 O 5 -CaO-MgO by sol-gel method. For the preparation of solutions based on the SiO 2 -P 2 O 5 -CaO-MgO system 2 compositions with different content of calcium and magnesium were chosen. Solution with MgO content of 20wt.% is suitable for film production up to 7 days (thickness of films obtained on silicon substrates 43.91±0.5). Solution with a magnesium content of 5 weight percent is available for creation of films only on the second day of its preparation (thickness of films obtained on silicon substrates 34.68±0.5). The formation of crystalline phases in synthesized materials occurs at 800°C. In sample Mg5 quartz SiO 2 and vitlokite Ca 2.589 Mg 0.411 (PO 4 ) 2 were identified. In sample Mg20, β-cristobalite SiO 2 and stentifieldite Mg 3 Ca 3 (PO 4 ) 4 were detected. Increasing MgO content in the system helps to accelerate the growth of particles on the surface of the samples when immersed in a solution of SBF, thereby increasing the bioactivity materials.