Synthesis and Characterization of Boron, Copper, and Zinc-Doped Hydroxyapatite by Sol-Gel Method: Research of Absorption and Thermal Behavior

In this study, hydroxyapatites reinforced with Boron, Copper and Zinc at different rates were produced using the sol-gel method. Different amounts of metal during the production of hydroxyapatite were used to observe the amount of crystallization and morphological differences in their structures. The characterization of the metal-doped HAp was carried out by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and Energy-dispersive X-ray spectroscopy (EDX). The data indicated that the Ca/P stoichiometric ratios of the samples varied between 1.71 and 2, so their morphologies were different from each other. The absorption behavior of novel metal-doped HAp samples was evaluated at room temperature by UV-vis spectroscopy. In the absorption spectra of the samples, absorption bands formed in similar regions. In addition, the thermal behavior of HAp samples was investigated using TG/DTA techniques. The results of the analysis showed that heat resistance of the new synthesized samples was quite high.


Introduction
Bone is formed of an inorganic roof and an inorganic matrix that supports and regulates this roof.
Inorganic roof of the bone is essentially formed of calcium and phosphate elements [1]. The smallest functional unit of the bone is called as osteon and when this structure is examined at electronmicroscopic level, it can be seen that the collagen bers with a diameter of one micrometer combine together and form the bone layers (lamels) and the channel system. On these collagen bers, hydroxyapatite crystals with a size of 100 nanometer are sequenced and form the microscopic structure of the bone [2]. Calcium hydroxyapatite (HA:Ca 10 (PO 4 ) 6 (OH) 2 ) and Tri-calcium phosphate (TCP:Ca 3 (PO 4 ) 2 ) develop the inorganic phases of human bones and teeth. Calcium phosphate-based bioceramic prosthesis have started to be used in the treatment of the bone and tooth diseases in rates increasing recently throughout the world [3][4][5][6]. In medicine, synthetic hydroxyapatites have been used commonly due to their properties such as biocompatibility, bioactivity, and osteoconductivity. Besides its numerous positive features, synthetic hydroxyapatites have low mechanical resistance and weak reactivity with the existing bones. In addition, they cannot be used in a wide range of applications for load bearing applications. In order to eliminate such disadvantages, metal ions with different properties and rates are added to the structure of hydroxyapatite (Al, Co, Fe, F, Cl, Sr, Si, Cu, Cr, Ba, K, Na, Mg, Zn, Mn, Cd, Se, Ni, Ti, and Y) [7][8][9][10][11][12][13].
Besides the use of HA as a powdered lling material in the body, it is also used in a sintered form. Signi cant changes occur in the mechanical property and stability of HA after the sintering process conducted to obtain resistance and a dense structure [14,15]. When HA is exposed to a thermal process between 900 and 1200 °C, changes occur in the Ca/P mol ratio in its structure. Slight deviations from Ca/P ratio of HA which is 1.67 may cause the formation of other calcium phosphate phases during these processes (alpha, beta-tricalcium phosphate, tetra calcium phosphate) [16]. Such different phases occurring after sintering are affected by the pH changes in the body.
Even though HA starts stable in the body without dissolution in the solutions with the pH values higher than 4.2 pH, as the pH value of TCP media increases, its stable structure is degraded and then absorbed by the body. This situation disadvantageous for TCP becomes an advantage if it is used as a synthetic bone material because bone growth occurs in the cavities forming due to dissolution after a while [17].
Today, various methods have been used in the production of synthetic HA. Examples can include PVD (physical vapor deposition), CVD (chemical vapor deposition), ion beam, laser, plasma spray, vacuum plasma, EPD (electrophoretic coating), HVOF (High velocity oxygen fuel thermal spray process), and solgel. All these methods have their own advantages and disadvantages. Sol-gel, one of the most preferred methods, is used to synthesize HAp due to high product purity at low temperatures and low cost [18][19][20][21][22][23][24][25][26][27][28]. In various studies, it is speci ed that the parameters such as temperature, period [29], acid addition rate, and precipitation drying rate [30] have signi cant effects on the particle size and morphology in the production of HA via chemical synthesis method. It is stated in the literature that some of the metal ions (Zn, Ag, Cu, Mg) that are added into HA during the production stage have an effect on the mechanical properties and antibacterial activity of the material produced [31,32]; especially the addition of boron (B) has an accelerating effect on the bone formation, and the apatite formation ability of B-doped HA samples is higher than those that do not contain B [33,34].
In this study, the synthesis of ve novel metal-doped hydroxyapatites by sol-gel method, using copper, zinc and boron as metal ions was investigated. The crystal structure and morphology of the obtained materials were shown using different spectroscopic methods. Also, thermal stabilities of novel materials were studied by using the TG-DTA techniques and their absorption behaviors were examined using UV-vis spectroscopy.  The presence of functional groups was analyzed by FTIR spectroscopy in the Perkin Elmer-Spectrum 100 FTIR instrument in the 400-4000 cm -1 range. 1 H-NMR spectra of schiff base monomers and polymers were recorded on an Agilent 400 MHz WB (Widebore) NMR instrument.
Rigaku SmartLab X-ray diffractometer, having a copper (Cu) X-ray tube, at 40 kV and 40 mA, with a wavelength of 1.544 Å and having Cu Kα X rays was used to characterize the crystallographic features of HAp powders. The surface morphology, elemental identi cation and quantitative composition of the prepared HAp were investigated by using a JEOL SEM 5300 scanning electron microscope. Thermogravimetric (TGA) and differential thermal analyses (DTA) were carried out at a heating rate of 20°C /min in the range of 0-1100 °C under nitrogen atmosphere using a TA Instrument SDT 2960.

Synthesis of schiff base monomer
The novel schiff base monomer was synthesized according to the following procedure:5-amino-1naphthol(3 mmol) and 3-formylfuran-5-boronic acid(3 mmol) were dissolved in ethanol/THF solvent mixture (5mL/2 mL). The reaction mixture was re uxed for 5 h at 80 °C. At the end of the process, the solution was evaporated and the solvents were separated from the formed schiff base. The obtained powder was washed with cold ethanol and diethyl ether and dried under vacuum over. MSB: 5-(((5-(dimethylboryl)furan-2-yl)methylene)amino)naphthalen-1-ol.

Preparation of novel metal-doped HAp
The sol-gel method was used to produce HAp and its production steps were realized as follows: to prepare the starting solution, 45.10 -2 moles of Ca (NO 3 ) 2 .4H 2 O, 2.10 -2 moles of KH2PO4 and 2.10 -2 moles of NaHCO were stirred in 20 mL of ethanol until a homogeneous mixture was obtained. The ethanol solution containing PSB, Cu(CH 3 CO 2 ) 2 and Zn(CH 3 CO 2 ) 2 in different rates was added into the current solution. Afterwards, the solution of P 4 O 10 (1.10 -2 mol, 5 mL H 2 O) was added into the homogeneous solution and stirring was continued for 10 minutes. The pH of the mixture containing all components was kept between 6-7 using ammonia. The pH adjusted solution was rst re uxed at room temperature for 1 hour and then at 50 °C for 1 hour. The resulting gel was subjected to aging process by being kept at room temperature for 24 hours. It was dried in the oven at 105 °C for 12 hours. In the last step, the dried mixture was calcined at 1100 °C for 3 hours ( Broad peaks formed in other groups in the structure. The peak observed between 1500 cm −1 and 1700 cm −1 formed as a result of overlapping of C=N and C=C stretching vibrations [35]. The single peak at 7.94 ppm proved the presence of the proton of the imine group (-CH = N-). Singlet and doublet peaks occurring in the range of 7.81-7.14 ppm were caused by naphthol and furan ring protons ( Figure 4). The naphthol proton peak formed very weakly in the spectrum of the polymer. This is because -OH group contributes in polymerization more than the benzene ring ( Figure 5). In the spectra of PSb, the two singlet peaks at 10.47 and 10.06 ppm belonged to the -OH proton of boric acid. The imine proton of PSb was observed in the range of 8.12-7.84 ppm as multiplet. In addition, the multiple, doublet and singlet peaks in the range 7.53-5.86 ppm belonged to aromatic protons [35].
Gel permeation chromatography (GPC) was used to determine the molecular weight (Mn and Mw) and polydispersity index (PDI) of the polymer (PSb  cm −1 indicated that there was CO 3 -2 absorbed in the HAp structure. In addition, very weak peaks around -2 (ν 2 ). This is because the presence of different metals in the structure changed the position of the CO 3 -2 group in the crystalline structure [36]. Two types of carbonate substitution are possible in hydroxyapatite. The rst is the direct substitution of OH by CO 3 -2 (A-type substitution CO 3 -2 ↔ 2 OH ) and the second is charge compensation, PO 4 3substituting tetrahedral group with CO 3 -2 (B-type substitution). The proof of the formation of both A and B type carbonate substitutions in the synthesized novel metal-doped hydroxyapatites is seen in their spectra [37].  Based on the XRD result of HAp/B 4 and HAp/B 5 , the peak intensities decreased and increased due to the change in metal content. Especially, the intensity of HAp/B 5 peaks decreased. Peaks proving the presence of HAp, β-TCP, and metal oxides (B, Cu, Zn) formed in the XRD data of both compounds.
The average particle size of HAp/B 1-5 powders was calculated using the "Scherrer" equation (Eq.1) D is Crystal size (nm), λ is the wavelength of CuKα1 radiation (1.5406 Å), β is the full width at half maximum for the diffraction peak under consideration, θ is half value of the diffraction angle of the most severe peak (2θ/2), and k is dimensionless shape factor. The particle size was calculated for highest intensity peak of HAp/B 1 , HAp/B 2 , HAp/B 3 , HAp/B 4 and HAp/B 5 , and the average of particle sizes was found to be 88 nm, 69 nm, 95 nm, 84 nm, and 70 nm, respectively.
On the other hand, the fraction of the crystalline phase of hydroxyapatite in the samples (Xc) was calculated using the (Eq.2) Xc = 100x((Ib-Va/b)/Ib) (2) where Xc is the fraction of crystalline phase, Ib is the highest intensity of diffraction peak, and Va/b is the intensity of the trough between the lowest and highest diffraction peaks. The speci c surface area was calculated with the formula(Eq.3) Ssp = 6 x 103 / d x ρth (3) where d is the average particle diameter and the theoretical density of the hydroxyapatite is ρth (HAP) = 3.16 g/cm 3 for spherical particles. Table 1 shows the calculated fraction of the crystalline phase and speci c surface area values. The crystal phase fraction of metal-doped hydroxyapatite powders is quite high as expected. The highest crystal phase fraction belonged to HAp/B 2 (99%) and the lowest crystal phase fraction belonged to HAp/B 5 (93%). The speci c area of metal-doped powders was between 19.98 and 27.51 m 2 /g and different speci c area values were obtained from each other due to the varying metal rates in the powders.

3.4.Morphological Investigations
Scanning electron microscopy images were used to show the surface morphology of the metal-doped HAp powders obtained and EDX spectra were taken for quantitative element analysis of the imaged regions. SEM images revealed that all powder samples had a different agglomerated spherical and granular morphology, due to the difference in metal content in the structure even when calcined at the same temperature. Differentiation of the morphology of the powders increased especially with the addition of Cu and Zn elements to their structures.
Macrostructure and microstructure SEM images obtained at different magni cations showed that HAp/B 1 and HAp/B 2 were quite different from HAp/B 3 , HAp/B 4 and HAp/B 5 in terms of roughness, pore size, geometry, and total porosity. Powder samples were compact, also had both micro and macroporosity and were not interconnected.
If the powder samples were compared in terms of porosity, it was observed that the best porous structure formed in HAp/B 1 and HAp/B 2 (Figure 10, 11). The presence of pores of different sizes in the morphology of these samples indicated that they can initiate an in ammatory process similar to the early phase of fracture healing when applied to the tissue, as in almost all arti cial bone tissue ceramics of mineral origin, depending on their porosity (17).
The similarity in the morphology of HAp/B 3 , HAp/B 4 and HAp/B 5 powders is that they created smooth structures with little pore structure ( Figure 12, 13, 14). A layered structure formed on the surface of all three powder samples. In addition, there were particles independent of the matrix structure and differently crystallized depression in their surfaces. The main reason for this formation was formation of metaloxygen bond in the hydroxy apatite structure, which is degraded by the effect of high temperature, regardless of the difference in metal rates. This bond indicated the presence of both metal oxides and metal-phosphate bonds.
Although the data obtained from the EDX analysis showed that the expected elements (Ca, P, O, C, B, Cu, Zn, K) were present in the prepared powder samples (Figure 15, 16). The data revealed that the Ca:P ratio was 1.86, 2, 1.90, 1.71 and 1.87 for HAp/B 1 , HAp/B 2 , HAp/B 3 , HAp/B 4 and HAp/B 5 , respectively, which was not very close to the ideal value of 1.67 normally associated with HAP. When HAp is exposed to high temperatures (900-1200 ο C), there is degradation at the Ca/P mol ratio in its structure. In particular, adding different metal ions to the hydroxyapatite structure causes this ratio to be different than expected. The deviations of HA from the Ca/P ratio of 1.67 may cause the formation of other calcium phosphate phases (alpha, beta-tricalcium phosphate, tetra calcium phosphate) during these processes.
In addition, Apatite has a chemical structure that allows it to be replaced with other ions. Due to this feature, the displacements in Ca +2 , PO 4 -3 or OH groups in its structure cause changes on the properties of its substance. EDX analysis results showed that the addition of B, Cu and Zn ions at different rates into the structures of newly prepared hydroxyapatite powders caused a different morphological structure than expected. This difference in powder structure is an expected situation and show parallelism with the main purpose of this study.
3.5.UV-Visible spectroscopy Figure 17 shows the UV-vis spectra of the novel HAp powders. Due to similar element contents, the samples formed absorption bands in same regions.

3.6.TGA analysis
The thermal properties of the prepared metal doped HAp powders were determined by thermogravimetric analysis (TGA) and differential thermal analysis (DTA) method in a nitrogen atmosphere heated at 20°C /min. in the range of 0 °C-1150 °C . Figure 18 and gure 19 show the TGA and DTA thermograms of prepared powders. From the TGA result of the powders, it can be seen that there was no phase transformation as a result of heating, thus indicated that the novel metal-doped hydroxyapatites had thermal stability at high temperatures.
The observed initial mass loss in the region of 20-200 °C can be due to the dehydroxylation of hydroxyapatite powders. There was no signi cant loss of mass for all powders during the thermal decomposition process. The main reason for this was B, Cu and Zn compounds in the structure of powders. Metals have high temperature resistance. Especially boron is very stable against temperature changes and today boron is used in the production of many materials that require thermal resistance, especially glass and ceramics. According to TGA data, the total mass loss of HAp/B 1 -HAp/B 5 between 20-1150 °C was 0.446%, %.433%, 2.426%, 3.235%, and 1.184%, respectively. The rst mass loss seen in the region of 200-700 °C is due to the removal of lattice bound water [53]. In this temperature range, the highest mass loss occurred at h4, while the lowest mass loss occurred at HAp/B 2 . Mass loss rates from small to large were 0.087%, 0.133%, 0.294%, 0.907%, and 1.237% for HAp/B 2 , HAp/B 1 , HAp/B 5 , HAp/B 3 , and HAp/B 4 , respectively.

Conclusions
In the rst stage of this study, a new polymer containing B was synthesized. This new compound contributed to the formation of both B 4 C and B 6 O in the structure of hydroxyapatite. In the second stage, new hydroxyapatite powders were obtained by sol-gel method using compounds containing Cu and Zn. Thus, ve compounds with different morphology were obtained during the formation of hydroxyapatite.
Especially the fact that HAp/B 1 and HAp/B 2 powders formed porous structures of different sizes showed that when applied to bone tissue, damaged tissue can heal in a short time.
Different spectroscopic methods were used for the characterization of the synthesized compounds and the data obtained proved that the targeted compounds were obtained. Especially, as a result of the thermogravimetric analysis, it was observed that their resistance to heat was high. HAp/B 4 had the highest heat resistance among the synthesized compounds. As a result of the heat treatment applied up to 1100 °C, only 2.21% of the structure was degraded and separated. The main reason for this is the different rates of metal content in the structure. Table   Table 1. The average particle size, fraction of crystalline phase and speci c surface area of HAp/B 1-5