Controlled Synthesis of Hydroxyapatite Nanomaterials Regulated by Di ﬀ erent Phosphorus Sources

: The morphology, nanostructures, and crystallinity of hydroxyapatite (HA) materials have signiﬁcant e ﬀ ects on their physicochemical properties and biomedical applications. However, the controllable synthesis of HA nanomaterials with various size and morphology using the same synthesis system, though desirable, has remained a challenge. In this work, we successfully synthesized HA nanomaterials with di ﬀ erent morphologies via a one-step solvothermal route in the same reaction system. By using (NaPO 3 ) 3 , Na 3 PO 4 · 12H 2 O and NaH 2 PO 4 · 2H 2 O as phosphorus sources, the growth of HA crystals was regulated, resulting in the transformation of HA nanomaterials morphology from nanorods to nanowires. Di ﬀ erent concentrations of PO 43 − and OH − ions in the reaction environments regulated by di ﬀ erent phosphates lead to di ﬀ erent nanostructures of HA crystals. Our work provides an instructive way to controllably fabricate HA nanomaterials with various morphologies and will widen the biomedical applications of HA materials.


Introduction
Hydroxyapatite (HA, Ca 10 (OH) 2 (PO 4 ) 6 ) has similar chemical composition with natural bone and teeth [1,2], showing excellent biological properties. The crystal structure of hydroxyapatite (HA) belongs to six fold space group (P6 3 /m, the highest symmetry) with unit-cell parameters of a = b = 9.421 nm and c = 6.884 nm [3]. HA has been widely used in biomedical fields such as hard tissue engineering, drug release, protein adsorption and bone cement filler [4][5][6][7]. Among various HA materials, nanophase HA is preferred because it can orchestrate cellular behavior by providing specific morphological and biological cues [8][9][10][11]. For example, ultralong HA nanowires with a high aspect ratio are very promising for adsorbent of organic pollutants, non-flammable paper, bone and tooth repair and bio-imaging [12][13][14][15].
Until now, the facile and controllable synthesis of HA nanomaterials with well-defined structure, size and morphology under the same synthesis system is desirable but remains highly challengeable.
Many synthetic methods have been employed to obtain HA nanowires, such as hydrothermal method [16,17], reverse micelles approach [18], hard template technique [19], micro-wave-assisted process [20] and sol-gel synthesis [21]. Phosphorus source is an important factor influencing the morphology of HA nanomaterials [22]. Recently, ultralong HA nanowires were solvothermally synthesized by using a series of phosphate sodium salts [23]. However, the synthesis time is relatively long and corresponding formation mechanism of HA nanowires needs further study.
In this study, we report a facile solvothermal method to synthesize aggregated HA nanorods, HA nanowires accompanied with flowerlike HA aggregates and ultralong HA nanowires under the same synthetic system by using three different types of phosphate sources. Effects of various phosphorus sources on the morphologies of HA nanomaterials are investigated and the morphological transition mechanism is proposed. This study provides an instructive way to realize the morphological transformation of HA nanomaterials without changing the synthetic system.
HA nanomaterials with different morphologies were prepared via a solvothermal synthesis. In the experiment, oleic acid (6.000 g) and anhydrous ethanol (6.000 g) were mixed on a magnetic stirrer (Zhengzhou, Henan, China) (300 r/min, 3 min). A total of 10 ml aqueous solutions of CaCl 2 (0.110 g) and 10 ml aqueous solutions of NaOH (0.500 g) were added to the above solution, respectively. After mixing well, the aqueous solution (5 ml) of phosphorus source ((NaPO 3 ) 3 :0.375 g; NaH 2 PO 4 ·2H 2 O:0.182 g; Na 3 PO 4 ·12H 2 O:0.443 g) was added dropwise into the above mixture, respectively. Then, the reactants were transferred into 50 ml stainless steel autoclaves and heated at 180 • C for 5 h. After being cooled down to room temperature, the resultants were collected and centrifugal cleaned thoroughly at 3000 rpm for 3 min with deionized water and anhydrous ethanol, and finally dried at 80 • C in an oven.
The phase composition of the products was measured by a Bruker D8 Advance X-ray diffractometer (XRD, Bruker Optik GmbH, Ettlingen, Germany) with a CuKα radiation source (λ = 1.5418 Å). The functional groups of the products were tested by a Fourier transform infrared spectrometer (FTIR, Bruker Tensor 27, Bruker Optik GmbH, Ettlingen, Germany). The morphology of the products was characterized by the field emission scanning electron microscope (FE-SEM, JSM-7610F, Tokyo, Honshu, Japan). To increase the conductivity of the samples, the products were sputter-coated with platinum before FE-SEM analysis.

Phase Analysis
XRD patterns of the as-synthesized products prepared by three different phosphorus sources, i.e., (NaPO 3 ) 3 , NaH 2 PO 4 ·2H 2 O and Na 3 PO 4 ·12H 2 O and the bar chart of standard HA (JCPDS No. 09-0432) are presented in Figure 1. The products show similar XRD patterns and all the main peaks are indexed to HA. The shift of the strongest peak from (211) to (300) is found when Na 3 PO 4 ·12H 2 O is used as the phosphorus source, suggesting preferential orientation growth direction along the c-axis of HA [24]. It is concluded that single-phase HA samples can be solvothermally synthesized by using CaCl 2 as the calcium source and different phosphates as the phosphorus source in mixed organic solvents of oleic acid and ethanol. The method is applicable for synthesizing HA materials using different kinds of phosphorus sources.

Functional Group Analysis
The FTIR tests were performed to characterize the functional groups of the samples. Figure 2ac represents the FTIR spectra of the as-synthesized samples by using (NaPO3)3, NaH2PO4·2H2O and Na3PO4·12H2O as the phosphorous source, respectively. As is shown in Figure 2, all the samples have similar patterns. The broad absorption peak at around 3420 cm −1 belongs to the adsorbed water in all the samples. The absorption peaks at around 1101, 1026, 962, 603, 560 and 472 cm −1 are the characteristic bands for PO4 3− group, and the peaks around 3571 and 633 cm −1 are attributed to the stretching and flexural modes of OH − , further confirming the formation of HA phase [25][26][27]. Moreover, the peaks at 3010, 2957, 2924, 2853, 1640, 1560, 1460 and 1413 cm −1 corresponding to the oleate group are originated from oleic acid molecules adsorbed on the surface of HA products [23].

Functional Group Analysis
The FTIR tests were performed to characterize the functional groups of the samples. Figure 2a-c represents the FTIR spectra of the as-synthesized samples by using (NaPO 3 ) 3 , NaH 2 PO 4 ·2H 2 O and Na 3 PO 4 ·12H 2 O as the phosphorous source, respectively. As is shown in Figure 2

Microstructural Characterization
FE-SEM images of the HA products prepared by different kinds of phosphorus sources were investigated. Figure 3 shows the morphology of the sample obtained by using (NaPO3)3 as a phosphorus source at different magnifications. It can be seen that the products are made up by a thick layer. From the magnified FE-SEM images (Figure 3c), the aggregates are self-assembled by nanorods. Figure 4 demonstrates the FE-SEM images of the sample synthesized by using

Microstructural Characterization
FE-SEM images of the HA products prepared by different kinds of phosphorus sources were investigated. Figure 3 shows the morphology of the sample obtained by using (NaPO 3 ) 3 as a phosphorus source at different magnifications. It can be seen that the products are made up by a thick layer. From the magnified FE-SEM images (Figure 3c), the aggregates are self-assembled by nanorods. Figure 4 demonstrates the FE-SEM images of the sample synthesized by using NaH 2 PO 4 ·2H 2 O as a phosphorus source. The dominant morphology of the products is nanowires with ultralong lengths. Except for nanowires, some flowerlike aggregates appear. The magnified FE-SEM image (Figure 4d) indicates that the flowerlike aggregates are self-assembled by nanorods. Figure 5 shows the morphology of the sample obtained by using Na 3 PO 4 ·12H 2 O as a phosphorus source. It is clear that the products consist of uniform nanowires. Besides, ultralong HA nanowires can interweave with each other and form a porous structure. From the FE-SEM images with low magnifications (Figure 5a,b), the HA nanowires are ultralong (lengths > several hundred micrometers) with, relatively, a high aspect ratio. These morphology changes among different samples when using different phosphorus sources are consistent with the XRD results.       From the above results, it is concluded that the morphology of synthesized HA gradually changes from agglomerated nanorods (Figure 3) to the coexistence of nanowires and flowerlike aggregates ( Figure 4) and then to well-defined nanowires ( Figure 5) under the control of different phosphates. The transition of such morphology confirms that phosphorus sources are crucial to the construction of HA nanomaterials. From the above results, it is concluded that the morphology of synthesized HA gradually changes from agglomerated nanorods (Figure 3) to the coexistence of nanowires and flowerlike aggregates ( Figure 4) and then to well-defined nanowires ( Figure 5) under the control of different phosphates. The transition of such morphology confirms that phosphorus sources are crucial to the construction of HA nanomaterials.

Formation Mechanism
We proposed a possible formation mechanism to explain the morphological transformation of HA nanomaterials by using different phosphorus sources in solvothermal process. The chemical reactions of different phosphates in the reaction system are listed as follows: The phosphorus sources used in the reaction environment exist as the forms of PO  4 3− and OHions in the reactions by using (NaPO 3 ) 3 as a phosphate are the lowest, since it takes more steps to obtain PO 4 3− ions. In this unique reaction system, oleic acid molecules can release Ca 2+ sustainably to regulate the preferential growth of HA crystal. According to the above chemical reactions and literature [28], the hydrolysis rate of three phosphates can be compared as follows: Na 3 PO 4 ·12H 2 O > NaH 2 PO 4 ·2H 2 O > (NaPO 3 ) 3 .
As is illustrated in Figure 6, the nucleation points of HA crystals multiply with the increase of PO 4 3− and OHions in the reaction environment. Figure 6A shows the self-assembly process of HA aggregated nanorods by using (NaPO 3 ) 3 as a phosphate. On one hand, a lower concentration of PO 4 3− and OH − ions is beneficial to the formation of HA nanorods. On the other hand, the P 3 O 10 5− ions generate by using (NaPO 3 ) 3 as a phosphate chelate with Ca 2+ in the reaction environment. The chelation hinders the growth of HA along the c axis, leading to the formation of HA nanorods. These newly formed nanorods are distributed freely at the bottom of the reactor. When the resultants are transferred to polar solvents, such as anhydrous ethanol, the alkyl chain molecules of oleic acid adsorbed on HA nanorods will make them self-assemble into regular arrangement patterns. The surface-induced instant self-assembly process is as follows. Oleic acid first adsorb on the surface of HA nanorods by the interaction between Ca 2+ ions of HA and carboxylic groups of oleic acid. Then the constituents of oleic acid in the final product diffuse into anhydrous ethanol accompanied with the washing process. HA nanorods can be separated from the solution due to incompatibility between the alkyl chain molecules of oleic acid and anhydrous ethanol. During the solvent diffusion process, oleic acid adsorbed on the surface of HA nanorods can modulate the self-assembly process of regular arrangement patterns by the "anhydrous ethanol−oleic acid" interaction, expelling HA nanorods to form regular arrangement patterns. When NaH 2 PO 4 ·2H 2 O is used as the phosphorus source, the concentrations of PO 4 3− and OH − ions in the reaction environment were higher than that with (NaPO 3 ) 3 ( Figure 6B). Therefore, the HA crystal nucleus was induced into short HA nanowires by oleic acid molecule. However, the concentrations of PO 4 3− and OH − ions in the reaction environment still lead to the formation HA nanorods. When the resultants are transferred to polar solvents, the short HA nanowires self-assemble to grow HA long nanowires under the action of the oleic acid alkyl chain molecules attached to them, while the HA nanorods self-assemble into flowerlike aggregates. As for the reaction of Na 3 PO 4 ·12H 2 O as the phosphate, more HA nuclei occur due to the high concentrations of PO 4 3− and OH − ions ( Figure 6C). Induced by oleic acid alkyl chain molecules, the HA nuclei grow rapidly into short HA nanowires as the reaction proceeded. Finally, ultralong HA nanowires with uniform morphology and good flexibility form when transferred to a polar solvent.

Conclusions
HA nanomaterials with different morphologies are synthesized through a one-step solvothermal route by using (NaPO3)3, Na3PO4·12H2O and NaH2PO4·2H2O as the phosphorus sources. The morphology of synthesized HA gradually changes from agglomerated nanorods to the coexistence of nanowires and flowerlike aggregates and then to well-defined nanowires under the control of different phosphates. The existence of phosphorus in the reactants leads to the morphology difference of HA nanomaterials. A possible formation mechanism of HA nanomaterials for the morphological transformation regulated by phosphates under solvothermal conditions is proposed. The strategy provides an instructive way to realize the controllable preparation of HA nanomaterials in the same reaction system and enlarge the biomedical applications of HA materials such as bone defect repair.

Conclusions
HA nanomaterials with different morphologies are synthesized through a one-step solvothermal route by using (NaPO 3 ) 3 , Na 3 PO 4 ·12H 2 O and NaH 2 PO 4 ·2H 2 O as the phosphorus sources. The morphology of synthesized HA gradually changes from agglomerated nanorods to the coexistence of nanowires and flowerlike aggregates and then to well-defined nanowires under the control of different phosphates. The existence of phosphorus in the reactants leads to the morphology difference of HA nanomaterials. A possible formation mechanism of HA nanomaterials for the morphological transformation regulated by phosphates under solvothermal conditions is proposed. The strategy provides an instructive way to realize the controllable preparation of HA nanomaterials in the same reaction system and enlarge the biomedical applications of HA materials such as bone defect repair.