Synthesis of High Hardness Hydroxyapatite Particles using Surfactant Assisted Hydrothermal Method

Hydroxyapatite (HA) crystals were synthesized from calcium acetate monohydrate and phosphoric acid using hydrothermal method. The various interactions of reactant concentrations (1 – 3 Molar), surfactant {aminotris (methylene phosphonic acid) [N(CH2PO3H2)3] (ATMP)} concentrations (0 100 ppm), and hydrothermal times (6 – 24 hours) were investigated and their effects on the mean diameter of the HA crystals were obtained using the Box-Behnken experimental statistical design. Results have shown that surfactant has no effect on the mean diameter of the crystals. On the other hand, the results revealed that time and reactant concentration are major parameters in changing the particle size of hydroxyapatite crystals. Without addition of the surfactant, well elongated crystals with high degree of crystallinity were synthesized. With addition of the surfactant, the agglomeration of HA particles as well as hardness of HA pressed discs are significantly increased. HA particles were ranged from 2.7 μm to 7.2 μm as crystal aggregates whereas the obtained crystallite sizes ranged from 17.3 nm to 30.3 nm.


Introduction
Hydroxyapatite (HA) is a complex salt of calcium and phosphorous with a stoichiometric composition of Ca10(PO4)6(OH)2 with a calcium to phosphorous ratio of 1.667. It has been studied intensively because of its presence in hard tissue materials such as the matrix of the bones, tooth dentin, and enamel. Usually, the HA content in the human body is around 65% [1]. Hydroxyapatite is important for its use in the biomedical field for artificial bone grafting because of its open porosity. Open porous HA allows a fast buildup of tissues and good osteoconduction. However, dense synthetic hydroxyapatite ceramic powders have exhibited low fracture toughness when compared with the values of human bones. Hydroxyapatite whiskers have generally exhibited high tensile properties because of their low dislocation density. With this view, needle-like crystals of hydroxyapatite have been synthesized for improving fracture toughness [2 -5].
The present investigation aimed to explore the effect of amino tris(methylenephosphonic acid) surfactant on HA morphology under hydrothermal conditions (i.e. at 140 o C) from phosphoric acid and calcium acetate monohydrate and to identify the optimum HA preparation conditions using Box Behnken experimental statistical design. Knowing that ATMP surfactant inhibits the crystallization of calcium sulfate dihydrate in an acidic medium, so, this research will explore the effect of ATMP on hydroxyapatite but at an alkaline pH of 9.0.

Hydrothermal synthesis
The Box Behnken design was used to develop an experimental procedure that would identify the main effects of varying factors (Table 1). From this data, optimal conditions could be obtained and conclusions based on statistical significance could be drawn. For these experiments hydrothermal time, concentration of reactants, and concentration of surfactant were varied throughout 15 experiments.
The reactants used were phosphoric acid and calcium acetate monohydrate from Fisher Company of 99.9% purity. They were added in stoichiometric amounts according to the following reaction: 6H3PO4 + 10Ca(C2H3O2)2. H2O Ca10(PO4)6(OH)2 + 20CH3COOH + 8H2O (1) According to the Box Behnken design in Table 1, the desired amounts of distilled water and surfactant were added to the reactor. The surfactant used was amino tris (methylenephosphonic acid) [N(CH2PO3H2)3] from Pfaltz & Bauer Incorporation (ATMP) of 0.18 % and has another name as Nitrilotrimethylphosphonic acid (amino tris). The purity of ATMP is 99.8 5%. Phosphoric acid of 99.9% purity and calcium acetate monohydrate were reacted in a Ca/P ratio of 1.667 without or with 50 ppm or 100 ppm of surfactant ( Table 1).
The calculated amounts of phosphoric acid and calcium acetate solutions were added to the reactor and allowed for one hour of reaction time with agitation. The resulting mixture was acidic (pH ranged from 2.4 to 3.4). After mixing for 1 hour, the solution was then adjusted to pH 9.0 + 0.1 using ammonium hydroxide. This pH is the optimum pH for precipitation of hydroxyapatite owing to its very low solubility at this pH. The mixture was then placed in a pressure vessel (half-filled) and put in the oven for the required time ranging from 6 hours to 24 hours at a constant temperature of 140 C. After, hydrothermal time, the resulting mixture was naturally cooled, filtered, and washed. The precipitated hydroxyapatite was dried at a temperature of 60 C for 24 hours. The dried samples were ground in a mortar to obtain fine crystals of hydroxyapatite, which are then pressed with a 25 mm die (ICL, Macro/ Micro KBr Die) used for isostatic pressing. The die is pre-lubricated with 1-2 drops of oleic acid and the obtained pellets are calcined and sintered. Sintering procedure was as the following: • Temperature was increased from room temperature to 800 C at a rate of 2 C per minute and maintained at 800 C for 2 hours. • Temperature is then raised from 800 C to 900 C at a rate of 2 C per minute and maintained at 900 C for 1 hour. • Temperature is then raised from 900 C to 1200 C at a rate of 2 C per minute and maintained at 1200 C for 1 hour.
• Finally, the temperature is then naturally decreased from 1200 C to room temperature.

Particle size distribution
Laser light scattering technique (Coulter LS 230, Beckman Coulter Corp., Miami, Fl) was utilized to identify the particle size distribution of the samples obtained. Samples were taken from a slurry of each Test. Each sample consisted of 8 mL of slurry dispersed in 50 ml of distilled water. All samples were then sonicated for 45 minutes and kept under constant agitation immediately before analysis. Then, the sample was poured inside the chamber of the Coulter instrument after flushing three times with distilled water.

Test
No.

Microstructure characterization
Hydroxyapatite samples were subjected to different characterization using X-ray diffraction analysis (BRUKER X-Ray Diffractometer), Crystallite size (Lc) of the hydroxyapatite phase was calculated using Scherrer's Equation No. 2: Where: k is the shape coefficient,  is the wavelength,  is the full width at half maximum (FWHM) of each phase, and  is the diffraction angle [15]. The hydroxyapatite particles were characterized by Scanning Electron Microscopy SEM (LEO 1455VP). Transmission Electron Microscope (TEM, JEOL-JM1230) was used for observation of the particle's morphology.

Hardness measurement
Vickers hardness of the obtained HA material is conducted after pressing and sintering the pellets. All the pellets were subjected to a Vickers microhardness tester (Micromet 3, Buehler LTD., Lake Bluff, IL). The pellets were indented on their polished surfaces. The Vicker's hardness of the pellets (HV) was calculated according to Equation (3), where, L is the indentation load in Newtons, 2a, the average diagonal length of the indentation in meters [30]. From 5 to 8 indentations were done on each specimen using a load of 1 kilogram with a 10-second loading time. Then average hardness value for each pellet was calculated.

Statistical experimental design tests
The obtained results of the 15 Tests of the experimental statistical Box-Behnken design with their experimental conditions are given in Table (1). The statistical Box-Behnken design program was applied to the obtained results. The resulting graphs are presented and discussed below showing general trends illustrating the most significant factors affecting the microhardness and the mean diameter of HA particles.

Effect of synthesis conditions on HA microhardness and mean diameter
All the experimental data with microhardness response are collected at the 3-D cubic and are given in Fig 4. This cubic diagram shows that the highest microhardness of about 397 to 537 Kg/mm 2 was achieved at a high level of surfactant concentration (100 ppm) at any level of other conditions. Also, all the experimental data with mean diameter response are collected at the 3-D cubic and are given in Fig. 5. This cubic diagram shows that a mean diameter of about 7.1 to 7.3 microns could be achieved at a high level of reactants concentration (3 M H3PO4), low level of hydrothermal time (6 hr), and with or without the addition of surfactant. With increasing reactant concentration, the HA mean diameter was increased. On the other hand, with increasing hydrothermal time, the HA mean diameter was decreased. The smallest mean diameter (3.09 -3.14 microns) can be obtained at a low level of

Effect of surfactant concentration and mean diameter on HA hardness
The effect of amino tris(methylenephosphonic acid) surfactant concentrations on hydroxyapatite hardness is given in Fig. 6. It is interesting that, with increasing surfactant doses, the HA hardness was increased whatever the other conditions. It is worth mentioning that, the hardness of human bone is 50 kg/mm 2 [31]. The obtained hardness values ranged from 108 to 494 kg/mm 2 which are higher than human bone hardness. The suggested mechanism for increasing the hardness by adding this surfactant is the formation of agglomerated nanoparticles which after sintering give high hardness and high shrinkage. On the other hand, tabular HA crystals will form without surfactant which gives low hardness. These findings are discussed in detail in item 3.8.
Particle size distribution was done for all the HA samples. Fig. 7 shows the particle size distribution of 5 samples (Tests 1 to 5). The mean diameter of the samples ranged from 2.7 µm to 7.2 µm (Table 1).
No correlation was found between HA mean diameter and hardness as shown in Fig. (8).

Characterization of crystallized HA powder
The precipitated powder was characterized using XRD, SEM, TEM, and Laser Size Distribution. X-ray diffraction pattern (Fig. 9) was conducted on the 15 samples. The precipitated phase was confirmed as hydroxyapatite [JCPDS # 00-009-0432]. The crystallite size ranged from 17.3 to 30.3 nm as shown in Table ( 1). The intensity of peaks at 2 Theta of 26 is increased from samples numbers 13, 14, and 15 (similar medium conditions) to sample number 12 (high reactant concentration with 100 ppm surfactant) to sample number 11 (high reactant concentration without surfactant) as shown in Fig. 9. Fig. 10 shows SEM photomicrographs of the crystallized hydroxyapatite powder samples (Tests 1 -4, 7, and 8). It is clear that the crystallized hydroxyapatite powders from all the Tests are agglomerated particles of different sizes. However, increasing reactant concentration (Tests 2 and 4) gave larger agglomerated particles compared with low concentration (Tests 1 and 3). To understand the effect of amino tris(methylenephosphonic acid) surfactant on the HA morphology, higher magnifications for the samples from Tests 7 and 8 are taken and given in Fig.  11. Test No. 7 was without surfactant and Test No. 8 was with 100 ppm surfactant. It was found that surfactant helps in the formation of agglomerated nanospheres. On the other hand, without surfactant, clusters from nanorods, nano tabular crystals, and nano rectangular crystals are formed. These crystals after sintering and pressing gave low hardness. Fig. 12 shows the TEM image of the crystallized hydroxyapatite powder sample (Tests 8). It is clear that the crystallized hydroxyapatite particles from Test 8 have 30 -80 nm size spherical shape particles. These particles are agglomerated to nanospheres with further agglomeration to micron spheres as shown from SEM.

Mechanism of hydroxyapatite precipitation and its morphology modification
Precipitation of hydroxyapatite is a very rapid crystallization. Crystallization is the result of three consequent stages: supersaturation, nucleation, and crystal growth [27]. The supersaturation stage occurs when the solution contains ordered aggregates of particles (clusters) of various sizes. The nucleus will grow in size after an aggregate exceeds the critical nucleus size [32]. So, supersaturation is essential for any crystallization because crystal nucleation must start from supersaturation. The pH of the solution was adjusted to 9 ± 0.1. It was reported that alkali treatment can convert brushite and tricalcium phosphate into hydroxyapatite [18 -22]. Fig. 13 shows the distribution of phosphate species at the hydrothermal temperature used (140 o C). It is clear that at pH 9, the predominant phosphate species is HPO4 2while PO4 3ions start to increase at pH 10. 50 % HPO4 2ions with 50 % PO4 3ions distribution is occurred at pH = 12.128. Almost similar results were reported in another paper [7] despite different reactants and conditions being applied for the synthesis of HA.    Regarding the chemicals used here, the solubility of calcium acetate is 34.7 g/100 mL of water. Calcium acetate, phosphoric acid, and water are mixed. The initial pH of the mixture is ranged from 2.4 to 3.4. Then, the pH of the mixture is adjusted by ammonium hydroxide to pH 9 ± 0.1. The effect of amino tris(methylenephosphonic acid) [ATMP] additive on hydroxyapatite crystal modification is attributed to sequestering (chelating of calcium ions). This leads to the agglomeration of fine aggregate (Fig. 11). However, without surfactant, well crystals (Tabular, Rod, and Cubic) are formed (Fig.  11). ATMP is a water-soluble (61 g/ 100 mL). In solution, it is partially hydrolyzed to anions with hydrophilic group (phosphonic groups and their counter ions). The phosphonic groups in the molecular chain have a strong tendency to attract positively charged ions or to be attached on positively charged surfaces. Therefore, the influences of ATMP on the agglomeration of nano aggregates could be attributed to two aspects: The negative phosphonic groups can chelate strongly the Ca 2+ in the solution, and then decrease the supersaturation by enhancing agglomeration rather than the formation of small nuclei. So, ATMP prevents scale formation in water systems.
On the other hand, the negative phosphonic groups can be attached to the positive crystal faces of HA. It is reported that hydroxyapatite has two different types of sites, called the P and C sites, in the crystal surface of the primitive unit cell. P sites (on the (a, b) crystal face) lack calcium ions or positive charge, and C sites (on the (a, c) or (b, c) crystal face) are rich in calcium ions or positive charge [33][34][35][36][37][38]. Therefore, the negatively charged phosphonic groups will be preferentially adsorbed onto the (a, c) or (b, c) crystal faces, namely, {100} and {010} facets of the nuclei, leading to modify the nucleation and crystal growth process of HA crystals.
As mentioned above, in the absence of ATMP, HA crystals grow naturally in the presence of enough free Ca 2+ ions in the solution. This means crystallization at a high supersaturation ratio, so high growth of tabular, rod, and cubic crystals are formed.
Chelating assisted hydrothermal synthesis of hydroxyapatite has been highly successful in obtaining agglomerated HA nanospheres. Surfactant poses the unique ability to regulate the agglomeration of hydroxyapatite particles. These particles after pressing and sintering gave high hardness compared to tabular, rod, and cubic crystals formed without surfactant.

Conclusion
Based on the results of the 15 hydrothermal treatment tests, it appears that the addition of amino tris(methylenephosphonic acid) [N(CH2PO3H2)3] (ATMP) surfactant helps for the formation of agglomerated hydroxyapatite particles with high hardness. Synthesis in the absence of surfactant resulted in the formation of hydroxyapatite of rod-like or tabular or cubic crystals. Therefore, at alkaline pH of 9.0, the surfactant does not affect the HA mean diameter but it increases the hardness significantly. Statistical experimental design analysis indicates that increasing the concentration of reactants led to the formation of large hydroxyapatite particles. The agglomerated hydroxyapatite particles are formed from agglomerated nanospheres of hydroxyapatite. Nanospheres hydroxyapatite was formed for the tests with a surfactant at different levels of reactant concentrations and hydrothermal times. HA particle sizes ranged from 2.7 µm to 7.2 µm, whereas the crystallite sizes ranged from 26 nm to 38 nm.