The effect of Sr and Mg substitutions on the mechanical properties and solubility of the uorapatite ceramics for biomedical applications

The ionic substitutions play important role in the modications of the biological apatites. Recently, the attention has been focused on the co-doping effects of additives on the functional properties of apatite based biomaterials. Under a research work for which the results are presented here, the dense samples of uorapatites: Ca 10 (PO 4 ) 6 F 2 and Ca 8 MgSr(PO 4 ) 6 F 2 were produced after sintering at a temperature of 1250 °C for 6 hours in air. The XRD, IR and Raman spectrometry results show a high crystallinity of the uorapatite and strontium-magnesium-doped uorapatites. The results demonstrate the stability of structural and mechanical properties of uorapatites after immersion tests in saline and buffer solutions. The durability of mechanical properties and biocompatibility of Ca 10 (PO 4 ) 6 F 2 and Ca 8 MgSr(PO 4 ) 6 F 2 uorapatites make these materials highly attractive for biomedical application. demonstrate stability of structural properties of


Introduction
Minerals and synthetic compounds with a structural type of apatite are widely used in many elds, including construction and electronic industries. Such materials also serve as catalysts and ion exchangers in the chemical industry. Apatites activated by rare earth elements are used as luminescent and laser materials. Moreover, apatite materials are considered as promising materials for immobilization of high-level waste (HLW) due to high chemical and radiation resistance and a wide range of structural iso-and heterovalent substitutions [1,2]. Some types of apatite materials have found wide application in orthopedics and dentistry due to the composition close to inorganic components of human bones and teeth [3][4][5].
Biological apatites, including hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 , HAp) have been widely used in the biomedical elds due to their bioactivity, biocompatibility and osteoconductive properties [6]. Recently, a large number of attempts have been made to the modi cation of biological apatites properties, such as biocompatibility, mechanical properties and solubility. Natural apatites contain various amounts of substitutions (F − , CO 3 −2 , Sr +2 , Mg +2 , Zn +2 ) [7][8][9]. Fluorine-substituted HAp (Ca 10 (PO 4 ) 6 (OH) x F 2-x , FHAp) and uorapatite (Ca 10 (PO 4 ) 6 F 2 , FAp) have low solubility and good biocompatibility [10,11]. The incorporation of uorine into apatite lattice makes the apatite structure more stabilized, a quite wellordered apatite structure was formed [12]. Fluorine replacement can favor the crystallization of calcium phosphate, improve the chemical stability and decrease the mineral dissolution [13,14]. In addition, substitution of OH − groups by F − results in a better protein adsorption and cell attachment [15]. Fluoride in saliva and blood plasma is necessary for dental and skeletal development and plays a very important role in stimulating the processes of proliferation and differentiation of bone cells. The osteoblast responses were improved through uoride incorporation [16].
Despite the fact, that apatites are widely proposed as biocompatible and osteoconductive materials, but they demonstrate some principal limitations for long-term clinical applications. A large number of studies have been made to enhance their mechanical properties, such as brittleness and mechanical strength. It is shown that the ionic substitutions in biological apatites play a key role in enhancing such functional properties as biocompatibility and mechanical strength [17][18][19].
Strontium (Sr), a trace element chemically close to calcium, is mainly incorporated into bone by two mechanisms: surface exchange or ionic substitution. Bone mineral consists of a poorly crystalline fraction made of apatite and calcium phosphate complexes. The strontium levels in bone are varied according to the bone structure. Furthermore, at the crystal level, higher Sr concentrations are observed in newly formed bone than in old bone [20].
There is experimental evidence that strontium induces pharmacological actions on bone metabolism [21,22]. The strontium ability to substitute calcium in the hydroxyapatite crystal lattice has been previously demonstrated [23]. Strontium has also been incorporated in the structure of new bioactive materials, and is used as a drug in the form of strontium ranelate to increase the densi cation of bone in osteoporotic patients. In vivo studies of Sr bioactive glasses [24] have shown that strontium has a dual function within bone remodeling. It is able to uncouple the process of bone resorption and bone formation by inhibiting osteoclasts and stimulating osteoblasts, respectively. Sr substituted calcium phosphate cements/ceramics were used in orthopedic, in lling bone defects [25]. The composite materials by adding 1 wt.% SrO to biogenic hydroxyapatite (HAp) or hydroxyapatite of biogenic origin -BHAp) have been proposed by Kuda et al [26]. It was found that BHAp/glass/SrO composite possessed a higher porosity and rate of dissolution in a physiologic solution. A bene cial effect of low doses of stable strontium in the treatment of osteoporosis age-related bone diseases was reported. The strontiuminduced increase of bone formation results in a better mechanical resistance of bones. The Sr-doped ceramics materials were considered for the development of coating or composite biomaterials to expand the range of biomedical applications [26].
In turn, magnesium (Mg) is one of the most important cationic substitutions for calcium in biological apatites. Dentin, enamel and bone contain 1.23, 0.44 and 0.72 wt.% of Mg, respectively. Over 100 enzymes require the presence of magnesium ions for their catalytic actions. These facts make Mg one of the essential elements for all living organisms [27]. Magnesium is closely relevant to mineralization of calci ed tissue and indirectly in uences mineral metabolism [28]. Magnesium de ciency affects all skeletal metabolism stages such as bone growth and bone fragility [29]. Fluorine-substituted HAp, FAp, and Mg-substituted apatites have received increasing attentions in the eld of biomedicine. Mg 2+ substituted FAp provides greater biocompatibility and better biological properties than pure FAp or HAp.
The formation and attachment of biomimetic Ca-P coatings in the Simulated Body Fluid (SBF) solution were strongly related to Mg 2+ content, where Mg-substitution improves the bioactivity of apatite in SBF [13]. In addition, Mg ion promotes bone-like apatite nucleation and growth on titanium surface in SBF solution and improves MC3T3-E1 (E1 type of MC3T3 is an osteoblast precursor cell line derived from Mus musculus of mouses calvaria) cell proliferation [30].
From the other side, recently, the attention has been focused on the co-doping effects of additives on the chemical dissolution behavior of biomaterials. Melt-derived Sr-containing polyphosphate glasses, doped with Mg and Ti were investigated by D. Weiss et al [31]. The inclusion of Mg and Ti was found to increase the bonding strength between phosphate chains resulting in a higher stiffness, better mechanical properties, and lower degradation rates in buffer solution. The properties of different melt-derived alkalifree phosphosilicate glass compositions co-doped with Zn 2+ and Sr 2+ ions were investigated in [32]. The compounds showed lower solubility as a result of the ionic eld strength associated with its constituent ions. There was a signi cant difference in the leaching of Zn 2+ and Sr 2+ ions in SBF and buffer solution, with a higher rate of release for Sr. Additionally, the substitution of SrO with CaO led to the partial replacement of Ca 2+ by Sr 2+ in the uorapatite and diopside crystal structures [33].
The effect of Sr and Mg substitutions in melt-derived glasses system of CaO-P 2 O 5 -Na 2 O was investigated by M. Stefanic et al [34]. The chemical durability of glasses in water was found to decrease with decreasing Sr content, and it was characterized by linear degradation and highly controllable pro les. The incorporation of Sr and Mg ions improve the solubility, bioactivity and mechanical properties of the glassceramics composite system. It was demonstrated that most Mg ions remained in the glass matrix and had a negative effect on the crystallization of apatite with a high Ca/P ratio. On the other side, the presence of Sr element was detected in all the deposited apatites, indicating that the introduced strontium was capable to substitute into the forming apatite nuclei and favor the formation of apatite crystallization.
In addition to biocompatible properties of calcium phosphate ceramics, the antibacterial effect of Sr and Mg substitution has not been well-understood and is an open issue of research. An inhibitory effect of Sr additives on various strains such as Escherichia Coli (E. coli) and Porphyromonas gingivalis (P. gingivalis) was reported in [35]. D. S. Brauer et al [36] shew that the bactericidal action of bone cements was increased via Sr substitution. The samples containing small amounts of Sr (2.5 mol%) reduced the number of bacteria (Streptococcus faecalis) up to one order of magnitude as compared to Sr-free samples. On the other hand, uorine ions are known to affect the mineralization and bone formation in vivo, resulting from their antibacterial effect. It can be concluded that Sr 2+ and Mg 2+ ions have a synergistic effect with F − ions in promoting the antibacterial activity.
Mg and Sr substituted phosphate ceramics and glasses are very attractive materials for the production of modern implants, prosthesis for orthopedic, dental surgery and scaffolds for tissue engineering applications [37]. It is well-known that the dissolution of ceramics in biological environment is complex and depends on numerous factors, such as phase composition, density, and surface parameters. Thus, a further focus on the effect of Sr and Mg co-doped ions on the structural and mechanical properties of calcium phosphate ceramics is of great interest. The aim of the present study was the evaluation of the effect of Sr and Mg ions substitutions on the mechanical properties and solubility of the uorapatite ceramic.

Materials And Methods
Fluorapatite Ca 10 (PO 4 ) 6 F 2 and strontium-magnesium-doped uorapatite Ca 8 MgSr(PO 4 ) 6 F 2 were prepared by chemical precipitation as previously presented in [2]. a. Sintering of uorapatite samples was carried out in air in a Nabertherm GmbH L5/13/B180 furnace. In order to produce uorapatite structures with higher density, the samples were sintered in the temperature range 1000-1250 °C for 6 hours in air.
b. Samples were formed by the method of cold double-sided axial pressing in a hydraulic press. c. Differential-thermal analysis and thermogravimetric analysis (DTA/TGA) were performed on a SDT Q600 V20.9 Build 20 Thermal analyzer. h. IR spectrometer IRS-29 (LOMO) was used to record absorption spectra in the IR range. Spectra were detected in the spectral range 4000-400 cm -1 (mid-infrared region).
i. Raman spectra were analyzed by a Raman spectroscopy method on confocal microscope (Renishaw inVia).
j. Mechanical properties tests of uorapatite samples for evaluation of Vickers hardness parameters and elastic modulus were performed by nanoindentation method on a NanoIndenter G200. The average values were evaluated as result of 10 prints at a depth of 1 μm on the surface of samples. Fracture toughness K 1c using Vickers indenter has been studied, as well.

Results And Analysis
The These results are in good agreement with the data of the previous study [38]. As can be seen in transparent electron microscopy (TEM) images (Fig.2), powders of Ca 10 (PO 4 ) 6 F 2 uorapatite synthesized by chemical precipitation method were agglomerated and mainly consisted from the nano crystalline particles with the average size of 20-30 nm (Fig. 2a). The Ca 8 MgSr(PO 4 ) 6 F 2 uorapatite powders produced by chemical precipitation method were also agglomerated with larger particles of the average size of 50-60 nm. (Fig. 2b).
As it is shown [39], ionic radius of the Sr 2+ (1.13 Å) is slightly bigger than of the Ca 2+ (0.99 Å) one. On the contrary, ionic radius of the Mg 2+ (0.65 Å) is signi cantly smaller than of Ca 2+ , and ionic substitution of the calcium by magnesium ions leads to noticeable decrease in lattice parameters of uorapatite. At present study, the substitutions of the Ca 2+ by the Sr 2+ and Mg 2+ ions in the synthesized Ca 10 (PO 4 ) 6 F 2 powders leaded to the increasing of uorapatite lattice parameters: a = 9.393Å; c = 6.894Å.
The differential thermal and thermogravimetric analysis (DTA/TGA) curves of powders Ca 10 (PO 4 ) 6 F 2 and Ca 8 MgSr(PO 4 ) 6 F 2 produced by chemical co-precipitation have a similar character (Fig. 3). The obtained data indicate the presence of two thermal effects. The rst one relates to the removal of adsorbed water at a temperature of ~ 60-110 °C. The second may be associated with the thermal decomposition of uorapatites by the reaction [40]: For uorapatite, the partial decomposition temperature was near 900 °C (Fig 3a). The partial decomposition temperature of uorapatite Ca 8 MgSr(PO 4 ) 6 F 2, however, decreases to 650 °C, when calcium is replaced by strontium and magnesium (Fig 3b).
The phase composition of sintered (1250 °C, 6 hours) strontium-magnesium-doped uorapatite sample was represented by the main phase Ca 8 MgSr(PO 4 ) 6 F 2 (Fig. 4). The weight content of the    Table 1. In contrast to uorapatite, a number of differences were observed in the spectrum of strontium-magnesium-doped uorapatite: -a characteristic peak of 725 cm -1 appears, corresponding to symmetric vibrations of the bridging bonds of the P-O-P diorthogroups. This indicates the association of phosphate tetrahedra; -the band disappears in the region of 960 cm -1 . This is caused by the degeneracy of the stretching vibrations of the PO 4 3ion due to a change in the coordination environment and symmetry of the PO 4 3ion due to the breaking of bonds between the phosphate tetrahedron and calcium ions; -signi cant reduction in the intensity of all bands for the spectrum associated with both the structure of uorapatite and adsorbed water.
There was detected, that the Sr and Mg doping of uorapatite results in reduction of the band intensity and band resolution corresponding to PO 4 −3 vibration modes. This fact has suggested that Ca is substituted by Sr and Mg in the uorapatite lattice. In a research related to Raman spectroscopic images of bones [42] it has been demonstrated that strontium can heterogeneously distribute in bone mineral, with a higher amount in newly formed bone tissue than in old bone tissue. Additionally, it is shown that the strontium incorporation does not make any signi cant change in the crystal lattice parameters. In this study, in accordance with the infrared spectroscopy spectra, the Raman spectra of uorapatite and strontium-magnesium-doped uorapatites exhibit internal vibrational modes for the group of PO 4 3-, see Fig. 8. Thus, the most intense line of the symmetric stretching vibration ν 1 (PO) at 961 cm -1 , which is a characteristic value for apatites [43], is clearly visible. According to [44], in the case of substitution of phosphate ions by carbonate ions, the phosphate line ν 1 appears in the range of 955-959 cm -1 . The line of symmetric stretching vibration ν 1 (PO) was detected at 961 cm -1 . This fact indicates the high crystallinity of uorapatite, which is con rmed by the XRD data (Fig. 4). In addition, the position of the ν 3 line (1038 cm -1 ) corresponding to the asymmetric P-O stretching vibration did not change. For uorapatite Ca 8 SrMg(PO 4 ) 6 F 2 , a decrease in the intensity of the ν 3 line and its fusion with the extended ν 1 line are observed (Fig. 8b). In contrast to the ν 1 and ν 3 lines, the remaining lines of the P-O deformation vibrations ν 2 (423→413 cm -1 ) and ν 4 (596→606 cm -1 ) are shifted towards lower frequencies when calcium is replaced by strontium and magnesium. In addition, on the Raman spectra of uorapatite and strontium-magnesium-containing uorapatite, a 1077 cm -1 line was observed. This line is corresponded to CO 3 2- tetrahedra. In [45] it is reported that the substitution of PO 4 3ions by CO 3 2ions does not cause any signi cant structural changes in the FAp structure that could be observed on the Raman spectra. These results are also in a good agreement with the IR analysis data.
Long-term immersion tests demonstrate the real behavior of the biomaterials in biological environment. In this regard, the solubility of the synthesized uorapatites in physiological media and the effect of the structural replacement of calcium by strontium and magnesium are of great interest.
The results of the solubility tests of uorapatites Ca 10 (PO 4 ) 6 F 2 , and Ca 8 MgSr(PO 4 ) 6 F 2 in saline solution are shown in Fig. 9a. A decrease in the degree of dissolubility of uorapatite in time was observed. These results also indicate a greater degree of solubility of strontium-magnesium substituted uorapatite in comparison to unsubstituted uorapatite. Fig. 9b shows the results of the solubility of the synthesized uorapatites in buffer solution. It should be mentioned that in the previous study [38] an increase of the weight losses and dissolution rates of strontium substituted uorapatite Ca 9 Sr(PO 4 ) 6 F 2 in comparison to unsubstituted uorapatite Ca 10 (PO 4 ) 6 F 2 samples was detected. Based on [2], this fact is associated with the presence of tricalcium phosphate Ca 3 (PO 4 ) 2 phase in the Ca 9 Sr(PO 4 ) 6 F 2 samples. It is known that tricalcium phosphate demonstrate greater solubility in comparison with uorapatite. In the contrast, it was reported in [46] that the strontium and calcium substitutions cause no principal change in the dissolution rate of phosphate glasses. Additionally, the effect of strontium bonding to a similar number of phosphate chains as calcium was found. The addition of Sr into the phosphate glass composition resulted in a decrease of the dissolution rate of the glass, thus suggesting an increase of the cross-linking between phosphate chains. At present study, the results demonstrate that in both saline and buffer solutions the solubility of strontium-magnesium substituted uorapatite is higher than uorapatite. It should be noted that in the buffer solution, the solubility of uorapatites was not much higher than the solubility in saline.
There is a strong correlation between the structure, mechanical properties and solubility of ceramics. The ceramics with higher density and crystallinity demonstrate better mechanical characteristics and biocompatibility [47]. The hardness (H) to the Young's modulus (E) ratio is the main parameter, which characterize the material deformation in relation to yielding [48]. H/E ratio plays an important role in identifying the mechanical behaviors and further brittle failure of ceramic materials and coatings [49].
Furthermore, fracture toughness (K 1c ) parameters demonstrate the ability of a material to resist against brittle failure and crack propagation. Nanoindentation method allows to make direct measuring of cracks created with a sharp diamond indenter [50, 51].
Taking the above-mentioned points into consideration, the main mechanical parameters of Ca 8 SrMg(PO 4 ) 6 F 2 and Ca 10 (PO 4 ) 6 F 2 uorapatites before and after immersion tests are presented in Table   2. The measured average value of Young's modulus on the surface of uorapatite slightly exceeds the value for strontium-magnesium doped uorapatite. After a solubility test in saline solution, the values of Young's modulus measured on the surface of both samples slightly decrease. On the contrary, microhardness and K 1c parameters for Ca 8 SrMg(PO 4 ) 6 F 2 samples decreased to a greater extent after immersion tests possibly due to the higher soluble tricalcium phosphate Ca 3 (PO 4 ) 2 phase content. Nevertheless, the dissolution of the synthesized uorapatites within 14 days occurs to a small extent and does not signi cantly affect their mechanical properties.
The surface of uorapatite samples after 14 days of immersion tests in saline solution was studied by Raman spectroscopy, SEM, and laser confocal microscopy.
An analysis of the Raman spectra of the surface of uorapatite samples after solubility tests also showed the presence of the main uorapatite lines ν 1 (P-O) at 965 cm -1 , see Fig. 10. These results indicate a strong stability of uorapatite and strontium-magnesium-doped uorapatite in saline solution at a temperature of 37 ºC. The presence of the ν 1 peak in the Raman spectrum con rms the stability of the crystal structure of uorapatite on the surface of the studied samples. After solubility tests for Ca 10 (PO 4 ) 6 F 2 uorapatite, a shift to the region of high frequencies of the line ν 3 (1038→1052 cm -1 ) and low frequencies of the line ν 2 (423→430 cm -1 ) were observed in (Fig. 10a). On the contrary, the line ν 4 was shifted to the low frequencies region (596 → 588 cm -1 ). At the same time, the position of the line corresponded to the vibrations of CO 3 2-(1077→1079 cm -1 ) was almost unchanged. For Ca 8 MgSr(PO 4 ) 6 F 2 uorapatite after immersion tests, shift of the lines of P-O deformation vibration ν 2 (413→425 cm -1 ) and ν 4 (606→628 cm -1 ) towards higher frequencies was detected (Fig. 10b).
The position of the line corresponding to the vibrations of CO 3 2- Fig. 11a and Fig. 11b. It is noticeable that the surface of the Ca 8 MgSr(PO 4 ) 6 F 2 sample underwent stronger dissolution as compared to Ca 10 (PO 4 ) 6 F 2 , see Fig. 11c and Fig. 11d.
According to the data of EDX analysis of surface after soaking tests, the main peaks of Ca, P, O, F, Sr and Mg corresponding to the elemental composition of uorapatites are identi ed in Fig.12.
The EDX spectra demonstrate some changes in the peak intensity after immersion tests. A noticeable decrease of the uorine content in the composition of the uorapatites samples was detected. C. Chaïrat et al [53] argue that the dissolution of apatite is primarily due to the relatively rapid removal of F and Ca from the contact surface. Thus, the destruction of uorapatite occurs due to the breaking of Ca-O bonds on the surface depleted of calcium and uorine.  2 was also presented mainly in the composition of strontium-magnesium doped uorapatite. The immersion tests in saline and buffer solutions indicated a greater degree of solubility of strontium-magnesium substituted uorapatite in comparison to unsubstituted uorapatite. According to scanning electron and laser confocal microscopy analysis, a pronounced surface dissolution of the strontium-magnesium doped uorapatite sample was observed. The mechanical parameters of uorapatites such as Young's modulus, hardness, and fracture toughness measured on the surface of the samples slightly decrease for both uorapatite and strontium-magnesium doped uorapatite after immersion tests.
Results demonstrate stability of structural properties of uorapatites after soaking in saline and buffer solutions. The durability of mechanical properties and biocompatibility of Ca 10 (PO 4 ) 6 F 2 and Ca 8 MgSr(PO 4 ) 6 F 2 uorapatites make these materials highly attractive for biomedical applications.

Declarations Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the nal version of the manuscript.