Structural characteristics and mechanisms of fluorapatite mechanochemical synthesis

Abstract This paper analyzes mechanisms of fluorapatite mechanochemical synthesis and its structural characteristics. Several studies of Jokanovic et al. published in appropriate journals and the book “Nanomedicine, the biggest challenge of the 21st century” are the base for this article. Characteristics of obtained materials show numerous biological advantages associated with the specific structural design of material during the process of synthesis. X-ray diffraction (XRD) and infrared spectroscopy with Fourier transform (FTIR) were used for studying the processes of fluorapatite synthesis.


INTRODUCTION
Fluorapatite (FA), chemical formula Ca 5 (PO 4 ) 3 F, or Ca 10 (PO 4 ) 6 F 2 is the most stable, least soluble, and the hardest calcium orthophos mineral (Mohs hardness scale 5). Such characteristics of fluoroapatite are associated to the specific position of F-ions in the center of the Ca 2 triangle and its crystal structure. The synthesis techniques are similar to those of hydroxyapatite, but it should be noted that synthesis of fluoroapatite involves the presence of F-ions, which are transmitted into synthesis through CaF, NaF or NH 4 F. Compared to hydroxyapatite (HA), which is Ca-deficient, there are no data to suggest Ca-deficiency of fluoroapatite. The chemical formula of fluorapatite is Ca 10 (PO 4 ) 6 (F,OH) 2 as the most frequent modification of OHions by Fions is not complete. Among all human calcified tissues, the greatest concentration of fluorapatite is found in bones, and the lowest in enamel. However, even where there is the largest concentration of fluorapatite, the amount of fluoride is usually reduced related to stoichiometric quantities. Due to its low solubility (degradation rate), it is rarely used as a bone substitute.
Due to mechanical stability, its solubility is reduced and proliferation of bone tissue is improved. Hydroxyapatite / fluorapatite (FHA / FA) has been used as clinical restorative material in the recent years [1,2]. In addition, FHA and HA / FA, are used in biomedicine as carriers of drugs and catalysts or absorbents [3,4].
Compared to HA [1], FHA / FA has better thermal and chemical stability [5,6]. When a certain number of OH groups in the HA matrix is replaced by Fions, thermal and chemical stability of FHA / FA ceramics increases signifi-cantly. Theoretically, the ratio F: OH ≥ 1 within the chain OH (in the FHA structure) would be sufficient to arrange HA crystals, stabilizing their structure due to alternating schedule of Fions among OHions.
In practice, materials that contain Fions are widely used for dental restorations as they prevent tooth decay and reduce bacterial activity in an acidic environment. In addition, Fions themselves favour mineralization and crystallization of calcium phosphate during bone formation [7]. Furthermore, in vitro studies FHA / FA have indicated its slow dissolution, better deposition layer as with hydroxyapatite, better adsorption of the protein [6][7][8], and similar or better cell attachment compared to pure HA [7,9] as well as improved activity of alkaline phosphatase [6].
It has also ben shown that the presence of fluoride affects the increase in quantity and quality of bone in body [5]. Fluoride ion is used to treat osteoporosis because bone mass increases with the application of Fions [9]. Fions also stimulate the activity of osteoblasts, both in vitro and in vivo. In addition, the mineral phase of enamel consists of HA (95 -97%) with from 0.04 to 0.07 wt. % Fluoride. A dose of about 1.5-4 mg of fluoride per day significantly reduces the risk of tooth decay [5]. In addition to FHA and FHA phase, materials like CaF 2 are also important in dentistry, because they can be used as reservoir of labile fluoride in caries prevention [10][11][12][13][14].
Some studies have shown that dual delivery system of (Fand Ca 2+ ions) is necessary to allow homogeneous nucleation and formation of very small crystals of CaF 2 in the mouth. These amounts are very efficient in increasing the deposition and retention of labile Fions in the mouth, while at the same time remineralization effect increases without consequent increasing of F-content [15][16][17][18][19][20][21][22][23]. According to the research of Jokanovic et al. [24] it was described for the first time not only a specific method of synthesis of fluorapatite, but also a synthesis of combined system encapsulated in surface-active substance polyethylene vinyl acetate / versatate, which is a potential source of labile CaF 2 phase. This is very important in order to maintain a balance of F ions content and to improve chemical and mechanical stability of the tooth.
For the synthesis of FHA / FA using precipitation, different methods are used like sol-gel, hydrolysis, hydrothermal method and solid phase reactions. They include appropriate ion exchange between the reactants that are used in the synthesis of FA [23][24][25][26]. Most chemical methods require very precise control of parameters of the synthesis process, product composition control and control of its characteristics, which is not so easy to achieve. Therefore, those methods are not suitable for the synthesis of FHA / FA on an industrial scale [27].
On the other hand, mechanochemical process is simple method that takes place in the solid state, allows synthesis of materials through the extremely efficient process of mixing different ion types due to shear forces, which using reduction of particle size and their alternating layers positioning improve thermodynamics and kinetic reactions between different solid substance precursors. In addition, compared to other above-mentioned processes, this method is more suitable regarding economic and technical sides because it enables mass production of nanocrystalline powders and high flexibility of process parameters [13].
The aim of this study was to present the method of synthesis of Nano powders fluorhydroxyapatite / fluorapatite using the method of mechanical alloying. Milling parameters such as speed of rotation, diameter, number of spheres, and weight ratio of the dust-spheres were constant, while the influence of milling time on phase composition was carefully defined. The kinetics and mechanism to obtain FHA / FA and other transitional phases were examined using XRD and FTIR spectroscopy.

MECHANISMS OF FLUORAPATITE SYNTHESIS
The mechanisms of fluorapatite synthesis are shown in the case of the most commonly used precursors such as calcium hydroxide Ca(OH) 2 , phosphorus pentoxide P 2 O 5 , and calcium fluoride CaF 2 (synthesis 1) and calcium hydroxide Ca(OH) 2 , phosphorus pentoxide P 2 O 5 and ammonium fluoride NH 4 F with the addition of surface-active substance vinyl acetate / versatate (EVA / AVV) (synthesis 2). Both mechanisms are carried out through the series of processing steps that can be analyzed using IR spectroscopy and X-ray diffraction [24]. It was noted that each phase is followed by certain degree of transformation of starting reactants in fluorhydroxyapatite with smaller segments of OHgroups and larger segments of Fions instead of OHgroups, until complete transformation of fluorapatite is finished. Generally, the reaction is carried out with an incomplete stoichiometry where x is thevalue that defines deviation from complete symmetry and can be found in the interval x 1  Based on X-ray diffraction, it was found that after only 1h of mechanochemical treatment, amorphization had occurred.
Due to extremely high concentration of mechanical strain on a very small contact surface (the contact that is realized in mutual globe collision or in globe collision with the surface of the inner lining) conditions are generated for the emergence of high shear stresses in a relatively small contact surface. Thus, the size of tension strain depends primarily on the diameter of spheres used in mechanochemical treatment (the size of the contact portion of a sphere indentation deformation in a crash) and the speed collision. Simultaneously, strain transfer leads to mechanical activation of the system and highly resilient flow that follows intense chemical and phase changes in the material (reaction shift, mixing ionic types, creating new phase, etc.). These changes can be such that material during the relaxation time partly suffers reversible deformation (highly resilient flow) or can be entirely irreversible when creeping material mechanism dominates.
The tension of critical deformation depends on the system exposure time to deformation (the number of sphere blows), in other words the number of pressure cycles, so that with time, the tension which provokes critical deformation demolition/formation of fissures and new areas, has less and less value, leading to larger amorphousness of the system. The process of mechanical activation in which water appears as a reaction product, is additionally accelerated by facilitating the transport of adequate ion types to places that correspond to the minimum of free energy of the system.
In this case, because of the exceptional hydrophilicity of P 2 O 5 , immediately upon its adding to other reactive substances, the process creates phosphorous acid (P 2 O 5 + 3H 2 O→2H 3 PO 4 ), which then reacts with Ca(OH) 2-2y (CO 3 ) y and creates Ca(HPO 4 ) 1-y (CO 3 ) y (carbonate calcium hydrogen phosphate).
After 4h of milling, the distinctive HPO 4 2start to vanish intensively and 5h after completely disappears, while the band on 963 cm -1 , appears (carbonate calcium efficienthydroxide fluorapatite) ( Figure 1). Simultaneously, during the whole process, CaF 2 dissociates and F ions that enter into reactionare created with calcium deficient hydroxide fluorapatite until the formation of its final chemical form. Finally, on previously mechanically treated samples during the period of 6 and 9 hours, and their afterwards thermic treatment on 1100°C, the bands belonging to CO 3 2disappear on 1420 and 1455 cm -1 . In samples mechanically treated for 6 hours one band ap-pears on 630 cm -1 indicating that exchange between fluor and hydroxide group was not completed. The band on 726 cm -1 that belong to F ions, is constantly present during the whole process demonstrating that F coordination is not significantly changed through the process of F transfer from CaF 2 into calcium hydroxide fluorapatite.
The rate of deprotonation of HPO 4 2-and ion exchange of OHand Fregulate the rate of formation of fluorapatite in all stages of the process. The process of dissociation of calcium fluorite occurs through the process of chemical etching of its particles, within the defects of the system (open surfaces, corrosion pits, dislocations, dislocation loops, vacancies), and tears Ca 2+ ions away from these places, leaving exposed Fions which are carried by water molecules andthen transported to places that correspond to a given fluctuating concentration gradient / concentration gradient of the local surface.
This indicates that it is realistic to assume that reactions in larger and smaller initial particles of the system take place in different ways and at different time intervals reach equilibrium conditions for the final reaction of fluorapatite formation. The reaction on the surface of large particles probably runs immediately after the begining of the mechanochemical treatment, while the cores that are still associated to given initial reactants remained in the depth of the particles. The morphology of the particles, which even in the remote stages of the treatment (4-6 hours) remains the same, testifies that the reactions in every particle / particle group advance individually. The reaction in smaller particles proceeds quickly, and in medium and large particles it progresses with full intensity along the newly created paths (new surface areas, the border of the crystallites / block mosaics, etc.), until the reaction of conversion of the calcium hydroxyfluorapatite into the fluorapatite is fully implemented, as it is shown on the refined XRD spectrum ( Figure 2). Figure 3 shows the nearest neighbours of Fion in the structure of carbonate fluorapatite. It is noted that there are three CaII 2+ located near Fion at a distance of 2.3 Å.  These CaII 2+ ions form the vertices of an equilateral triangle with Fion in the center. There are three CaII 2+ , P 5+ , and O 2ions in the second coordination sphere, which mutually form the vertices of triangles. The distance between Fand P 5+ is 3.6 Å, and between P 5+ and O 2-(1) is 3.9 Å. There are O 2-(3) ions above and below the plane containing Fand CaII 2+ . The oxygen ions occupy the vertices of a dodecahedron. The distance between Fand O 2-(3) ions is 3.1 Å, whereas between P 5+ and O 2-(3) ions it is 1.5 Å. Figure 2 shows a fragment of the crystal lattice compared with the unit cell. According to the research of Pandaet al.CaII 2+ has larger atomic size compared toCaI 2+ . When OHions are substituted with F -, there is greater distortion in the structure due to the larger size of the ionic radius of F -. At the end of the mechanochemical synthesis process, Fion occupies large space in the center of the latticeforming a stable fluorapatite structure.
According to the research of Jokanovicet al. [24] (OH -, F -), in addition to the three types of [OH -], the chain of apatite also contains the fourth type with different vibrational energies. It is observed in this study that if the criteria for displacement of free vibration OHis taken as the criteria for quantifying the changes of OHwith F -, then it is indicated that about 50% of OHgroups are modified with F -, while the system, with almost completely changed OHgroups with F -(for pure fluorapatite), provides the value of the wave number of 758 cm -1 ( Figure 4 and Table 1).

THE METHOD OF SYNTHESIZING WITH ADDITIONAL LOW-TEMPERATURE THERMAL TREATMENT
Another method of mechanochemical synthesis using the precursorsCa(OH) 2 , P 2 O 5, NH 4 F and surfactant of vinyl acetate / versatate, shows that mechanochemical process only can not form fluorapatite. That is why it is necessary to carry out an additional low-temperature treatment.
FTIR method ( Figure 5) proved to be the most suitable method for monitoring the synthesis. In order to obtain complete picture of phase transitions that occur in materials during mechanochemical and low-temperature treatment, the method of X-ray diffraction [23] can be used in addition to FTIR method.
XRD spectra of samples ( Figure 6) show very intense peaks of portlandite (P), while β-Ca 2 P 2 O 7 (CP) peaks are also visible. In addition, CaCO 3 (C) and CaF 2 (CF) peaks are strongly emphasized. All the characteristic diffraction peaks for FA are almost negligible, and some of them absent [29].
After 5 minutes of milling, the most intense peaks belong to Ca(OH) 2 , and typical peaks for β-Ca 2 P 2 O 7 , CaF 2 and CaCO 3 are clearly visible. The peak corresponding to Ca(OH) 2 is clearly visible and shows low rate of reaction for forming fluorapatite. Consequently, the amount of synthesized FA in the mixture is negligible.   There were identified changes in the structure from amorphous to crystalline, for all thermally treated samples. The typical peaks of FA confirmed transformation that took place in almost all samples (Figure 7). The amount of residual CaCO 3 and CaF 2 was still significant only in the sample milled for 5 minutes. The emphasized peaks of these phases indicate that parts of the samples remained unchanged, despite the high energy involved in the mechanochemical treatment during the preparation of the precursors mixture ( Figure 7a).
As shown in Figure 7 a-c, clearly emphasized and sharp peaks typical for FA are present as a result of an adequate mixing of samples for 2 hours and particularly milling for 8 hours. The sample milled for 5 minutes shows that the transformation of the reaction mixture in flourapatite was only partial, despite the thermal treatment at 550°C for 3 hours. In addition to FA peaks, there are also CaCO 3 and CaF 2 peaks. This proves that even though the inside of the micelle of a surfactant contains the components of the building blocks of precursors phase that can be easily transformed into the pure fluorapatite, they still remain unchanged.
On the contrary, the mixture that was milled for at least 2 hours and additionally thermally treated for 3 hours is completely transformed into the pure fluorapatite. Similar was for a sample that was milled for 8 hours. The peak corresponding to FA only moves towards the greater angles of diffraction. This means that the content of OHgroups was reduced during the milling of the sample and that FA finally became predominant phase (possibly mixed with a small amount of hydroxyapatite).

REACTION MECHANISM
During 5 minutes and 2 hours of milling, the reactions in which β-Ca 2 P 2 O 7 and CaF 2 are formed are dominant, while Ca(OH) 2 part remains unchanged. This has a strong influence on the synthesis rate of FA, which is very slow and cannot be completed only by milling (even after 8 hours of milling). This low rate comes from a very slow diffusion and rearrangement of certain ions that are necessary for FA formation. The exchange and incorporation of Ca 2+ ions in β-Ca 2 (1-x) P 2(1-x) O 7(1-x) and Ca (1-x) F 2(1-x) pre-formed cells is strongly inhibited by the presence of EVA/EVV. Therefore, in order to provide bigger reaction rate of formation of FA, crystal structure of β-Ca 2 (1-x) P 2(1-x) O 7(1-x) and Ca (1-x) F 2(1-x) must firstly be transformed to amorphous by additional milling (2 -8 hours). This procedure provides good mixing, which reduces the diffusion paths of different ions.
The second stage of the formation of FA started after low temperature treatment of the samples at 550°C for 3 hours. In this step, according to certain researches, the reactions can be initiated on the surface of the dominant phase β-Ca 2 P 2 O 7 through the surface diffusion of additional Ca 2+ and Fions in its volume. According to the diffraction peaks, it is evident that the process of formation of FA during these thermal treatments is very intense. The only exception was a sample milled for 5 minutes. Despite conducted thermal treatment, short milling time was not  It is important to emphasize that, no matter how long the samples were milled; this reaction wouldn't be possible without the thermal treatment. With the extended milling time (up to 3 hours or more), the system became amorphous. The reaction did not progress even during the longest milling time (8 hours), which was confirmed by XRD. The progress was observed only in the amorphous samples (milled for at least 2 hours), which were subjected to subsequent thermal treatment. The mechanism of the process, which took place during milling, was possibly significantly activated by the water present in Ca(OH) 2. The explanation provided by some researchers shows that smaller Ca(OH) 2 particles, during milling under the influence of shearing forces, tend to grow, causing further disintegration [29][30][31], so that the exchange of different ion species becomes more efficient.
During milling, the distortion of Ca 2+ -O polyhedra is much more prominent in comparison with P 5+ -O tetrahedra. The distortion led to displacement of cations from the center of its coordination sphere. It has a strong impact on the diffusion rate of the remaining amount of Ca 2+ and Fions and consequent destruction of β-Ca 2 P 2 O 7 during milling. Thus, the "empty" space in β-Ca 2 P 2 O 7 is increasinglyfilled with these ions, untilβ-Ca 2 P 2 O 7 cell is completely destroyed. It should be noted that the capacity of dissolution of Ca 2+ and Fions within β-Ca 2 P 2 O 7 is very high. Accordingly, the smaller size of CaF 2 , and especially Ca(OH) 2 crystallites is suitable for further propagation reaction of the formation of FA. Although crystallization of amorphous phase cannot be achieved through milling, the distribution of ions provides very rapid crystallization of the samples in FA in the next step of a very low thermal treatment (at 550 °C for 3 hours).
Therefore, it seems that there is enough space within β-Ca 2 P 2 O 7 structure for F-anions to be placed in large gaps, and within the network of calcium and phosphate ions. In addition to Fions, very small CaF 2 nanoparticles are placed randomly in the gaps within β-Ca 2 P 2 O 7 lattices, which produce significant changes in its symmetry, causing the corresponding chemical changes responsible for the transformation of mixture into fluorapatite during the next thermal treatment. This treatment can significantly accelerate the processes of diffusion, causing degradation of EVA/EVV micellar cages and supporting small ion redistribution by distortion in β-Ca 2 P 2 O 7 structure induced by shear forces, until the final transformation of the mixture into fluorapatite.

CONCLUSION
Mechanochemical process of fluorapatite synthesis is based on the use of two kinds of precursors: calcium hydroxide, phosphorus pentoxide and calcium fluoride, or calcium hydroxide, phosphorus pentoxide and ammonium fluoride with the addition of surfactant vinyl acetate/verstat. On the basis of XRD and FTIR analysis it was observed that fluorapatite has significant advantages in comparison with hydroxyapatite. These benefits are related to its greater stability, lower solubility and especially better protection against cavities. Poslednjih godina fluorohidroksiapatit/fluorapatit (FHA/ FA) koristi se u kliničkoj restauraciji, jer mu je zahvaljajući mehaničkoj stabilnosti smanjena rastvorljivost i unapređena proliferacija ćelija koštanog tkiva [1,2]. Pored toga, HA i FHA/ FA se koriste u biomedicini kao nosači lekova ikatalizatora i adsorbensi [3,4].