Investigation of Inclusion States of Silicate and Carbonate Ions in Hydroxyapatite Particles Prepared under the Presence of Sodium Silicate

Biological hydroxyapatite (HA) contains the different minor ions which favour its bio-reactivity in vivo. In this study, the preparation of HA particles containing both silicate and carbonate ions under the presence of sodium silicate was investigated, and the physicochemical properties were evaluated according to the contents and states of silicate and carbonate ions. The increment in the silicate ion reduced the crystallinity and expanded the crystalline size along with a-axis. Solid-state 29Si–NMR spectra indicated the increase in the adsorption of oligomeric silicate species on the HA particle surfaces in addition to the substitution state of silicate ions, suggesting the occurrence of the surface coating of silicates on the surfaces. The possible states of carbonate and silicate ions at the HA surfaces will provide the bioactivity.


Introduction
The similarity in composition and structure of hydroxyapatite (HA, Ca 10 (PO 4 ) 6 (OH) 2 ) to the inorganic phase of bones and teeth and its biocompatibility allow it to be used for biomedical applications [1,2]. The low-crystalline HA is suitable for ion substitution in both calcium (Ca 2+ ) and phosphate (PO 4 3− ) ions due to its labile crystal structure [3]. The biological HA usually contains the different minor ions such as Na + , K + , Mg 2+ , Sr 2+ , F − , Cl − , carbonate (CO 3 2− ), and silicate (SiO 4 4− ) ions [4][5][6]. The substitution or inclusion of these minor ions in HA changes its morphology, crystalline structure and physicochemical properties [7], leading to the enhanced bioactivity [8,9]. Among them, the important ions in the synthetic HA for biological applications are the SiO 4 4− and CO 3 2− ions since the mineral bone contains substantial amounts of the ions [10]. In biological bones, the contents of CO 3 2− and SiO 4 4− ions are approximately 4-8 wt% [8,11] and 0.4-0.5 wt% [12,13], respectively. The SiO 4 4− ions (e.g., silicate ion) are indispensable in the early stages of bone and cartilage formation, since they promote the mineralization, contributing to proper osteointegration and helping to bond the implanted material with the biological bone [9,14]. Thus, the incorporation of silicate ions in the HA structure can significantly improve the biological reactivity of HA [15]. On the other hand, the PO 4 3− ions of HA can be substituted by the CO 3 2− ions, generating the B-type carbonated hydroxyapatite (CHA) [16,17]. The B-type CHA has greater solubility, subserving the Ca 2+ and PO 4 3− ion concentration, which favours the osteointegration [18,19]. Furthermore, CHA enhances the osteoclast resorption allowing new bone formation [20,21]. Therefore, the understanding of the inclusion of silicate and carbonate ions in HA is the basis for effective control of the bioactivity. Although diverse studies have been carried out on the substitution of carbonate and silicate ions in HA [22], the mechanism of substitution and/or inclusion in low-crystalline HA has not been completely clarified [23][24][25].
The aim of this study is to control the simultaneous incorporation of SiO 4 4− and CO 3 2− ions into HA to obtain low-crystalline HA particles (SiHA) under the presence of sodium silicate in order to increase the HA bioreactivity. The preparation conditions are similar to the physiological conditions used to obtain the particles that are close to the biological HA. The mechanism of substitution with SiO 4 4− and CO 3 2− ions in the HA particles was investigated using the Fourier transform infrared spectrometer, X-ray diffraction, field emission scanning electron microscope, and solid-state 29 Si-NMR spectra recorded by dipolar decoupled magic-angle spinning.
The characterization was performed by using Fourier transform infrared (FT-IR) spectrometer, wavelength dispersive X-ray fluorescence (XRF) spectrometer, X-ray diffraction (XRD), field emission scanning electron microscope (FE-SEM), and solid-state 29 Si-nuclear magnetic resonance recorded by dipolar decoupled magic-angle spinning ( 29 Si-NMR DD-MAS). Based on the FT-IR spectra methodology, the quantification of CO 3 2− ions (wt%) was performed by the relative absorbance area ratio of v 1 CO 3 2− (1570-1330 cm −1 ) to v 1 v 3 PO 4 3− (~900-1230 cm −1 ) [26]. According to Scherer's equation (K = 0.89), crystalline size along the a-axis (D 300 ) was calculated from the half width of the D 300 diffraction patterns in the XRD. In the solid state 29 Si-NMR spectra, the chemical shift was adjusted by referencing the peak position of tetramethylsilane, and was evaluated by the spectral separation technique based on the peaks of Q 0 (-72 ± 2 ppm) due to isolated silicon unit, Q 1 (-74 ± 2 ppm) due to one Si-O-Si and three Si-OH bonds [27] and Q 2 (-90 ± 2 ppm) due to two Si-O-Si and two Si-OH bonds [28].

Results and Discussion
The Si concentrations obtained by XRF were 0.4, 0.7, 3.3. 5.0 and 6.9 wt% for 0.8SiHA, 1.5SiHA, 4.0SiHA, 6.0SiHA and 8.0SiHA. As shown in Figure 1a, the Ca/P molar ratio in the SiHA particles increased with the increase in feed Si concentration, suggesting the substitutions of PO 4 3− with SiO 4 4− ions in the HA. Due to the ion substitution, the amount of PO 4 3− ions decreased, causing an increase in the Ca/P molar ratio [18]. On the other hand, the Ca/(Si + P) molar ratio only decreased slightly with the increase in the feeding of Si, indicating the inclusion of SiO 4 4− ions in the HA. The FT-IR spectra of the SiHA particles ( Figure S1) show the stretching vibration band of v1(PO4 3− ) ions at 960 cm −1 . The band assigned to Si-OH vibrational modes of SiO4 4− ions appears at 890 cm −1 . The P-O stretching vibration bands of PO4 3− ions (1100, 1045 cm −1 ) and the Si-O-Si asymmetric bands of SiO4 4− ions (1040-1200 cm −1 ) are located very close, which makes interpretation difficult. The bands at around 1470, 1550 cm −1 correspond to the asymmetric stretching mode of v3CO3 2− ions. The band at ~3570 cm −1 is assigned to the OH − stretching mode [29][30][31][32]. It was observed that the increase in Si concentration caused an increase in the intensity of the Si-OH band and a decrease in the intensity of the v1(PO4 3− ) band, corroborating the inclusion of SiO4 4− ions by the substitution of PO4 3− ions. Furthermore, increasing the Si concentration increased the intensity of the CO3 2− bands, indicating the simultaneous substitution of PO4 3− ions with SiO4 4− and CO3 2− ions in the HA to form SiHA. This can be described by the possible formula (e.g., 2PO4 3− →SiO4 4− + CO3 2− ) [33]. The incorporation of SiO4 4− ions caused a decrease in the intensity of the OH − band until it almost disappeared in 8.0SiHA, implying the loss of OH − ions to compensate for the excess negative charges generated by the substitution of the PO4 3− ions with more negative ions (SiO4 4− ). Using the Kröger-Vink notation [34], the charge compensation mechanism [35] can be expressed as follows: Therefore, the increase in Si concentration effectively caused an increase in the content of CO3 2− ions, as shown in Figure 1b, indicating that the presence of SiO4 4− ions induced the inclusion of CO3 2− ions in HA. The incorporation of SiO4 4− and CO3 2− ions into HA increased the defects in the HA structure, resulting in more vacancies of OH − (V OH · ) due to more substitutions of PO4 3− ions. The simultaneous substitution of the PO4 3− ions in the HA with SiO4 4− and CO3 2− ions can be described as follows [13,36] Figure 2 shows the XRD patterns of HA and SiHA particles at the different Si concentrations. All the patterns were ascribed to a HA (Ca10(PO4)6(OH)2, ICDD: 00-009-0432). It was observed that the increase in Si concentration resulted in a decrease in crystallinity, which was caused by the defects and the formation of • due to the substitution of PO4 3− ions with SiO4 4− and CO3 2− ions in the HA structure. The calculated D300 crystalline sizes along with the a-axis of all the SiHA samples were smaller than the HA (Figure 2 (inset)) due to the incorporation of SiO4 4− and CO3 2− ions into the HA structure [37]. It was observed that the value of D300 first decreased (0.8SiHA) and then began to increase with increasing the Si concentration. Especially, 8.0SiHA had the highest value of D300, suggesting that the substitution of PO4 3− ions with SiO4 4− ions would occur predominantly, since 2− ions. The band at~3570 cm −1 is assigned to the OH − stretching mode [29][30][31][32]. It was observed that the increase in Si concentration caused an increase in the intensity of the Si-OH band and a decrease in the intensity of the v 1 (PO 4 3− ) band, corroborating the inclusion of SiO 4 4− ions by the substitution of PO 4 3− ions. Furthermore, increasing the Si concentration increased the intensity of the CO 3 2− bands, indicating the simultaneous substitution of PO 4 3− ions with SiO 4 4− and CO 3 2− ions in the HA to form SiHA. This can be described by the possible formula (e.g., 2PO 4 3− →SiO 4 4− + CO 3 2− ) [33]. The incorporation of SiO 4 4− ions caused a decrease in the intensity of the OH − band until it almost disappeared in 8.0SiHA, implying the loss of OH − ions to compensate for the excess negative charges generated by the substitution of the PO 4 3− ions with more negative ions (SiO 4 4− ). Using the Kröger-Vink notation [34], the charge compensation mechanism [35] can be expressed as follows: Therefore, the increase in Si concentration effectively caused an increase in the content of CO 3 2− ions, as shown in Figure 1b, indicating that the presence of SiO 4 4− ions induced the inclusion of CO 3 2− ions in HA. The incorporation of SiO 4 4− and CO 3 2− ions into HA increased the defects in the HA structure, resulting in more vacancies of OH − (V · OH ) due to more substitutions of PO 4 3− ions. The simultaneous substitution of the PO 4 3− ions in the HA with SiO 4 4− and CO 3 2− ions can be described as follows [13,36] Figure 2 shows the XRD patterns of HA and SiHA particles at the different Si concentrations. All the patterns were ascribed to a HA (Ca 10 (PO 4 ) 6 (OH) 2 , ICDD: 00-009-0432). It was observed that the increase in Si concentration resulted in a decrease in crystallinity, which was caused by the defects and the formation of V · OH due to the substitution of PO 4 3− ions with SiO 4 4− and CO 3 2− ions in the HA structure. The calculated D 300 crystalline sizes along with the a-axis of all the SiHA samples were smaller than the HA (Figure 2 (inset)) due to the incorporation of SiO 4 4− and CO 3 2− ions into the HA structure [37]. It was observed that the value of D 300 first decreased (0.8SiHA) and then began to increase with increasing the Si concentration. Especially, 8.0SiHA had the highest value of D 300 , suggesting that the substitution of PO 4 3− ions with SiO 4 4− ions would occur predominantly, since the radius of the Si 4+ (0.042 nm) is greater than that of P 5+ (0.035 nm), and the bond length of the Si-O bond (0.16 nm) is greater than that of the P-O bond (0.15 nm) [20,38], which must result in a higher D 300 value. These results are consistent with the crystal sizes observed in FE-SEM images ( Figure S2). In the images, all the SiHA particles exhibit needle-like shapes with the smaller sizes than HA, and theaggregation forms were observed. the radius of the Si 4+ (0.042 nm) is greater than that of P 5+ (0.035 nm), and the bond length of the Si-O bond (0.16 nm) is greater than that of the P-O bond (0.15 nm) [20,38], which must result in a higher D300 value. These results are consistent with the crystal sizes observed in FE-SEM images ( Figure S2). In the images, all the SiHA particles exhibit needlelike shapes with the smaller sizes than HA, and theaggregation forms were observed.  Figure 3a shows the deconvolution curves and fitting results of the spectra of the SiHA particles provided by their Q0~Q2 components. At the lower Si concentration (0.8SiHA and 1.5SiHA), only Q0 peak was observed. However, at the higher Si concentration (4.0SiHA, 6.0SiHA and 8.0SiHA), the Q1 and Q2 peaks appeared. The Q0 peak of the SiO4 tetrahedron corresponded to the stretching Si-O band in the FT-IR spectra of SiHA. The possible inclusion mechanism of the silicates ions into HA structure is shown in Figure 3b. It was suggested that the substitution with SiO4 4− ions in the HA structures was related to the inclusion of CO3 2− ions, which was proposed by the reaction mechanism based on the substitution of phosphate ions in Figure 3(b-1). Polymerized silicate chains (e.g., oligomeric silicates) due to Q1 and Q2 peaks appeared at the Si concentration of 3.32 wt%, suggesting that the unsubstituted SiO4 4− ions began to adsorb on the SiHA with the polymerization and condensation reactions. At the Si concentrations of 5.03 and 6.92 wt%, the intensities of Q1 and Q2 increased, suggesting that the unsubstituted SiO4 4− ions started to form oligomeric fragments and adsorbed on the SiHA. These results indicated that the oligomeric fragments are larger in the presence of more unsubstituted SiO4 4− ions as shown in Figure 3(b-2).   Figure 3a shows the deconvolution curves and fitting results of the spectra of the SiHA particles provided by their Q 0~Q2 components. At the lower Si concentration (0.8SiHA and 1.5SiHA), only Q 0 peak was observed. However, at the higher Si concentration (4.0SiHA, 6.0SiHA and 8.0SiHA), the Q 1 and Q 2 peaks appeared. The Q 0 peak of the SiO 4 tetrahedron corresponded to the stretching Si-O band in the FT-IR spectra of SiHA. The possible inclusion mechanism of the silicates ions into HA structure is shown in Figure 3b. It was suggested that the substitution with SiO 4 4− ions in the HA structures was related to the inclusion of CO 3 2− ions, which was proposed by the reaction mechanism based on the substitution of phosphate ions in Figure 3(b-1). Polymerized silicate chains (e.g., oligomeric silicates) due to Q 1 and Q 2 peaks appeared at the Si concentration of 3.32 wt%, suggesting that the unsubstituted SiO 4 4− ions began to adsorb on the SiHA with the polymerization and condensation reactions. At the Si concentrations of 5.03 and 6.92 wt%, the intensities of Q 1 and Q 2 increased, suggesting that the unsubstituted SiO 4 4− ions started to form oligomeric fragments and adsorbed on the SiHA. These results indicated that the oligomeric fragments are larger in the presence of more unsubstituted SiO 4 4− ions as shown in Figure 3

Conclusions
The inclusion of both silicate and carbonate ions was successfully achieved in the presence of sodium silicate. The content of silicate ions increased with increasing Si feed. The silicate ions promoted the inclusion of carbonate ions in the HA. The substitution of phosphate ions with silicate and carbonate ions into the HA structure produced defects and hydroxyl vacancies, generating a loss in crystallinity and smaller crystalline sizes. The substitution of phosphate ions was mainly dominated with silicate ions in the structure, inducing the particle growth along with the a-axis. The results of solid-state 29 Si-NMR DD-MAS demonstrated that at the lower Si feed, only ion substitution has occurred. At the higher Si content, the remaining silicate ions with the saturation of substitution formed the (poly)silicate species (i.e., oligomeric silicates). It was observed that these (poly)silicate species have a strong tendency to adsorb to the HA surfaces, modulating the crystal growth of the SiHA. Thus, the solid state 29 Si-NMR spectra indicated that upon reaching saturation in the substitution of phosphate ions with silicate ions, the remaining silicate ions polymerized to form (poly)silicate species adsorbed on the surface of the HA, suggesting the appearance of the silicate surface layer on the SiHA surfaces. There is a possibility to control the silicate ion states, the inclusion amount of carbonate ions in SiHA and its crystalline size by the feed concentration of sodium silicate. The incorporation of silicate and carbonate ions into the HA structure is expected to enhance the bioactivity of the SiHA particles.

Conclusions
The inclusion of both silicate and carbonate ions was successfully achieved in the presence of sodium silicate. The content of silicate ions increased with increasing Si feed. The silicate ions promoted the inclusion of carbonate ions in the HA. The substitution of phosphate ions with silicate and carbonate ions into the HA structure produced defects and hydroxyl vacancies, generating a loss in crystallinity and smaller crystalline sizes. The substitution of phosphate ions was mainly dominated with silicate ions in the structure, inducing the particle growth along with the a-axis. The results of solid-state 29 Si-NMR DD-MAS demonstrated that at the lower Si feed, only ion substitution has occurred. At the higher Si content, the remaining silicate ions with the saturation of substitution formed the (poly)silicate species (i.e., oligomeric silicates). It was observed that these (poly)silicate species have a strong tendency to adsorb to the HA surfaces, modulating the crystal growth of the SiHA. Thus, the solid state 29 Si-NMR spectra indicated that upon reaching saturation in the substitution of phosphate ions with silicate ions, the remaining silicate ions polymerized to form (poly)silicate species adsorbed on the surface of the HA, suggesting the appearance of the silicate surface layer on the SiHA surfaces. There is a possibility to control the silicate ion states, the inclusion amount of carbonate ions in SiHA and its crystalline size by the feed concentration of sodium silicate. The incorporation of silicate and carbonate ions into the HA structure is expected to enhance the bioactivity of the SiHA particles.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biomimetics7020040/s1, Table S1: Added amounts of the reagents in the synthesis of the SiHA particles; Figure S1: FT-IR spectra of the HA and SiHA particles; Figure  S2: FE−SEM images of the HA and SiHA particles.