Preparation of nanostructured hydroxyapatite in organic solvents for clinical applications.
Abstract: Composite materials consisting of Hydroxyapatite (HAP) nano-crystals and biocompatible polymers have been widely used in orthopedic and dental application. These composite can provide both ease of use and superior mechanical properties of polymers, osteoconductivity and bioactivity of HAP. The formation of HAP in different organic solvents and water was investigated to fabricate such composites at moderate temperatures. HAP nano-sized crystals with high degree of crystallinity were formed at room temperature by rapid addition of [(N[H.sub.4]).sub.2] HP[O.sub.4] solution into Ca[(N[O.sub.3]).sub.2.4[H.sub.2]O] solution, filtration and subsequent drying at 40 [degrees]C. The effect of different organic solvents, addition mode and temperature on the characteristics of HAP was examined. The HAP powders were characterized using Fourier transform infrared spectrometer (FTIR), X-ray diffraction (XRD), and Transmission electron microscopy (TEM). The HAP crystal shape was governed by the type of solvent: fine regular spheres and rods were formed from organic solvents with lower dielectric constants while irregular particles were prepared using water with higher dielectric constant. The synthesized HAP rod crystals were between (20-100) nm length and (2-6) nm thickness. All the FTIR and XRD profiles of HAP fabricated from ethanol were in good agreement with the FTIR and XRD spectrums of commercial HAP. It is therefore feasible to fabricate HAP uniform nano-particles in organic solvents at room temperature, which would be of benefit for the fabrication of polymer-HAP composites for biomedical applications.
Subject: Biomedical engineering (Mechanical properties)
Hydroxylapatite (Mechanical properties)
Powders (Mechanical properties)
Authors: Alobeedallah, Hadeel
Ellis, Jeffrey L.
Rohanizadeh, Ramin
Coster, Hans
Dehghani, Fariba
Pub Date: 01/01/2011
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: Jan, 2011 Source Volume: 25 Source Issue: 1
Accession Number: 308129316
Full Text: Introduction

Bone consists of approximately 8 wt% water, 22 wt% protein and 70 wt% mineral [1]. The mineral component of bone is a form of calcium phosphate known as hydroxyapatite (HAP), with the chemical formula [Ca.sub.10][[(P[O.sub.4]).sub.6][(OH).sub.2] and the hexagonal crystalline structure. HAP accounts for about 65 wt % of bone and provides most of its strength and stiffness. HAP crystals in bone are generally in the form of needle-like crystals in the nanometer-sized range of 5-20 nm width by 60 nm length [2]. The synthesis of nano-structured HAP is of considerable interest due to its broad applications in orthopedic, dental, and drug delivery application [3-7]. Studies have shown that nano-sized HAP has a high surface energy, which may improve its mechanical properties and allow for a faster implant surface turnover [8].

In bone tissues, HAP particles are associated with extracellular matrix proteins such as collagen [9]. The combination of HAP crystals and collagen fibers contributes to superior mechanical properties of bone and compensates for HAP low formability and slow degradation rate. Thus it would be ideal to combine HAP with a polymer matrix to take advantage of the properties of each material while minimizing shortcomings [10-14].

Synthesis of HAP

The techniques that have been developed to synthesis HAP are listed in Table 1. The widely used methods include wet chemical precipitation (aqueous based solvent) and sol gel (organic based solvent) [15].

Most of the current methods such as wet chemical precipitation for the synthesis of HAP involves post-heat treatment such as sintering at 1100 [degrees]C and final calcinations (hydrothermal treatment) [16-17]

The heat treatment results in crystal growth and an increase in coarseness of the microstructure. The use of high temperature, however, precludes the preparation of in situ HAP /biodegradable polymer composites as many polymers degrade at such high temperatures.

Wet chemical precipitation is generally performed in water by mixing solutions of calcium hydroxide and phosphoric acid as follows [2].

10Ca[(OH).sub.2] + [H.sub.3]P[O.sub.4] [right arrow] [Ca.sub.10][(P[O.sub.4]).sub.6][(OH).sub.2] + 18[H.sub.2]O

Calcium nitrate Ca[(N[O.sub.3]).sub.2] x 4[H.sub.2]O and diammonium hydrogen phosphate ((NH4)2HPO4) have been used to prepare HAP using wet chemical precipitation at pH values between 10 and 12. In such preparations the particle size varies as a function of the rate of addition of diammonium hydrogen phosphate to calcium nitrate, temperature, and stirring time. Jarcho and Yubao [26] reported that HAP with particle sizes less than 100 nm could be acquired in wet chemical precipitation by stirring the solution for 24 hrs.

Several attempts have been reported using sol gel method for the preparation of HAP. In the sol gel method an organic solvent is used as an alternative to water for the reaction between the reactants. This has the advantages of faster rate of precipitation and solvent evaporation compared with aqueous based systems. In addition, the degree of agglomeration between nano-sized particles is less in organic solvent than aqueous based system [14]. This may be related to lower capillary forces between particles and weaker hydrogen bonding in ethanol system compared to that in water.

Kuriakose prepared nanocrystalline HAP from organic solvent systems using the sol gel method to promote the characteristics of HAP [16]. The sol gel method can be used to synthesize a pure nano crystalline HAP at 85[degrees]C at alkaline pH followed by post processing by sintering at 1200[degrees]C. The particle size and shape of HAP fabricated by this method was not reported. Yanji Zang used a very similar method where aqueous solution of [(N[H.sub.4]).sub.2]HP[O.sub.4] was added to Ca[(N[O.sub.3]).sub.2] ethanol solution. Single phase nanocrystalline HAP was precipitated, while the morphology was changed from spherical to rod shape when the temperature was increased from 40[degrees]C to 80[degrees]C in the slow addition mode. However, at 80 [degrees]C, spherical particles were formed by the rapid addition mode [17].

Daiwon Choi [27] reported a room temperature process for the fabrication of HAP using organic solvent such as tetrahydrofuran. Spherical HAP particles were formed with diameter between 1 and 10 mm using tetrahydrfuran. The aim of this study was therefore to investigate the feasibility of fabricating nano-size and needle-like shape HAP crystals at low temperature, mimicking the morphology and size of bone apatite crystals.

Materials and Methods

Commercially available starting materials were purchased from suppliers and used without furher purifications as follows: diammonium hydrogen phosphate [(N[H.sub.4]).sub.2]HP[O.sub.4]) (Merck supply), calcium nitrate tetrahydrate [(Ca(N[O.sub.3]).sub.2] x 4[H.sub.2]O) (Stem-Supply), ammonia solution (NH4OH) (Sigma-Aldrich), tetrahydrofuran (THF) (SigmaAldrich) and absolute (anhydrous) ethanol (SigmaAldrich).


Preparation of HAP

The procedure for preparing HAP is schematically illustrated in Fig.1. To synthesize HAP powder 0.5 M [(N[H.sub.4]).sub.2]HP[O.sub.4] solution was prepared and added with continuous stirring to a solution of 0.5 M Ca[(N[O.sub.3]).sub.2] x 4[H.sub.2]O to maintain 1.6 Ca/P ratio. Solvents such as water, ethanol and THF were used. When an organic solvent was used (THF, Ethanol) the solutions were prepared first by dissolving each reactant in 5ml water and the pH was adjusted to within 10 [] 12 by addition of ammonia solution. The solutions were then diluted with the organic solvent. Two reaction modes were used: i) slow addition, which involved the addition of [(N[H.sub.4]).sub.2]HP[O.sub.4] solution drop wise with a rate of 5 ml/min; and ii) the rapid addition, which involved the addition of the [(N[H.sub.4]).sub.2]HP[O.sub.4] solution all at once. Experiments were conducted at various temperatures between 20[degrees]C and 70[degrees]C and various reaction times between 2 hr to 24 hr. The reaction was allowed to proceed for 24 hr with constant stirring, after which the solution was filtered and washed, and then the precipitated materials were collected and kept in the oven at 40[degrees] C overnight.


FTIR spectroscopy

Attenuated total reflection Fourier transform infrared (ATRFTIR) spectroscopy was used for the characterization of synthesized HAP powders. The spectrums were collected in the range of 500-4000 [cm.sup.-1] using a Varian 2000, Scimitar series, instrument.

X ray diffraction

X ray diffraction patterns were recorded using a Siemens D5000 diffractometer with CuKa radiation (e = 0.154nm).

The diffractometer was operated at 40 kV and 30mA at a 2e range of 10-60 degrees employing a step size of 0.05[degrees] and a 2 seconds exposure.

Transition electron microscopy (TEM)

A Phillips CM120 Bio twin microscope operating at 120 kV was employed to study the morphologies of the prepared samples. The samples for TEM were prepared by dispersing a small amount of the sample in ethanol and sonication for 30 minutes. A few drops of the resultant suspension were dropped on to a copper grid.

Results and Discussion

X ray diffraction (XRD)

Single phase pure crystalline HAP was precipitated under all conditions used in this study as corroborated by XRD analysis shown in Fig.2. The XRD profiles of the samples were in good agreement with the XRD patterns of a HAP standard available, JCPDS (09-0432). The XRD patterns possessed a strong peak at around 31.8[degrees] corresponding to (211) planes of HAP crystalline structure [27]. No other characteristics peaks corresponding to other calcium-phosphate phases or impurities were observed. The presence of additional calcium-phosphate phases such as dicalcium phosphate dihydrate (DCPD) would have changed the dissolution characteristic, thereby the bioactivity of precipitated materials.

The XRD patterns in Fig. 2 show the effects of the three different factors on crystal structure characteristics of the prepared HAP: addition mode in ethanol; reaction temperature in both water and ethanol; and finally the effect of the solvent.

The effects of the addition rate of calcium solution into phosphate solution on the size of the HAP crystals was studied using P.Scherrer approach [28] and the smoothed XRD patterns in Fig. 3. The rate of addition of Ca solution had a significant impact on the particle size and the degree of crysrallinty of the HAP powder. The rapid rate of addition used to prepare (sample b in Fig. 3) resulted in sharp patterns at (002) and (211) peaks, indicating that the crystallites were larger and/or better crystallinty than those fabricated at the same conditions but using the slow addition rate (sample a). The broad peaks of HAP particles in the sample a (in Fig. 3) resulted from the slow rate of addition of the Ca solution and gave HAP crystallites a nanocrystalline nature.

The degree of crystallinty of the HAP fabricated using various conditions was also compared using the smoothed XRD patterns shown in Fig. 3. The XRD patterns of the HAP fabricated from a water-based system (sample f) had a minimum intensity at peak 211 (118 Lin counts), corresponding to a poor degree of crystallinty in comparison with those prepared at room temperature using THF and ethanol. Sample (b) prepared at room temperature using the rapid addition mode in ethanol exhibited the sharpest peak at 211 plan as shown in Fig. 3 (b), indicating superior crystallinty.

The crystallite size of the eight different samples was compared using Scherer equation as follows:

t = (.9[lambda]/Bcos[[theta].sub.B]), B = [[theta].sub.1] - [[theta].sub.2]

Where, [e.sub.1] is peak start angle, [e.sub.2] is peak end angle, [e.sub.B] is maximum intensity angle, e is wavelength (Cu Ka = 1.5406 nanometer), and t is thickness (diameter) of the crystallites, [e.sub.1] [e.sub.2] and [e.sub.B] were detected using the analytical X-ray program EVA MFC Application 3 provided by Bruker analytical X-ray systems. The results are summarized in Table 3.

The results in Table 3 demonstrate that for the same solvent and the same addition mode the particle size of HAP was enhanced by increasing the temperature of the process. Table 3 shows that as the reaction temperature was changed from room temperature (sample a) to 40[degrees]C (sample c) and finally to 70[degrees]C (sample d) De decreased, indicating an increase in the particle size. The same behavior was observed for the samples prepared using water at 40[degrees]C and 70[degrees]C. This effect can be elaborated by an increase in the crystal growth rate caused by increasing the temperature. It was also found that the particles prepared at room temperature using the slow addition mode such as sample []a[] were smaller than sample []b[] that was processed under the same conditions using the rapid addition mode.


FTIR Spectroscopic analysis

The results of the FTIR analysis are shown in Fig. 4. All FTIR profiles were in good agreement with the FTIR spectrum of commercial HAP [27-28]. These results once again confirmed the formation of pure form HAP under the various conditions used in the experiments. The P[O.sub.4.sup.-3] bands were detected at wave numbers of 1091, 1071, 1036, 602 and 564.9 [cm.sup.-1]. The hydroxyl bending bands of HAP were identified at around 3560 [cm.sup.-1] and 624 [cm.sup.-1]. The bands attributed to absorbed water were also found at 870 [cm.sup.-1]. The absorption bands that appeared at 1450 and 1380 cm-1 were indicative of the presence of the carbonate ion which could have resulted from dissolved (ambient) C[O.sub.2] during the crystallization process. The amount of dissolved ambient C[O.sub.2] could be reduced by either performing the operations at lower ambient C[O.sub.2] pressures or by subsequent sintering of the material at 800[degrees]C. It should be mentioned that at low temperature C[O.sub.2] replace P[O.sub.4] in HAP crystalline structure. Carbonate substitution has a critical role in dissolution properties of HAP and it has been shown that this substitution will increase the solubility and reduce the crystal size of apatite [29].


Transmission Electron microscopy Analysis (TEM)

The TEM images in Fig. 5 demonstrate that the HAP powders fabricated from all the solvents used in this study were in the sub-micron to nano-size range with all having longest dimentions of 100 nm. The particles formed from ethanol solutions were rod-like shape crystals. Those synthesized in THF had very fine spherical crystals and the sample synthesized in water had irregular to spherical shape particles. The addition rate had negligible effect on the shape of HAP particles prepared at room temperature using ethanol and both sample (a) and (b) showed very fine rod-shape crystals. However, rapid addition of the solutions resulted in the formation of larger particles (Fig. 5). The larger crystals may have been formed due to the lower number of crystal nucleation sites because of rapid addition of Ca in P[O.sub.4]. Increasing the temperature resulted also in enhancing the particle size, which may be due to faster growth rate at higher temperatures. The results in Table 5 were consistent with the data acquired from Scherrer model in Table 3, both demonstrating that increasing the temperature and rapid addition mode promoted the size of particles.

Our results for the precipitation of HAP from THF solution at ambient temperature are in good agreement with Daiwon Choi who prepared agglomerated spherical particles (10im) of HAP at room temperature from a THF solution [27].

Our hypothesis is when an organic solvent is used; the electrostatic forces between the ions that form the HAP crystals are higher than in water as predicted by Coulomb's law. The electrostatic forces are inversely proportional to the dielectric constant (relative permittivity) of the medium. The electrostatic forces will, therefore, be considerably smaller in a solvent such as water (e = 78) compared to ethanol (e = 24). Stronger and longer range interactions between the ions in organic solvents would be expected to achieve faster reaction and nucleation rates and hence smaller particles with regular morphology. There might be also a correlation between dielectric constant of the solution and particle morphology. Decreasing the dielectric constant of the solution increased the intermolecular interaction between the particles and a greater tendency to form regular crystalline macrostructure with smaller sizes. This hypothesis can explain the formation of regular rod-shape crystal in ethanol (e = 24) and very fine regular spheres in THF solution (e = 7.2). The HAP crystal size in THF and ethanol solution was smaller than the irregular shape and larger particles in the samples prepared from water, which has relatively higher dielectric constant compared to THF and ethanol.



Single phase and relatively highly crystalline nano-size and rode-like HAP particles were synthesized at room temperature. The XRD and FTIR results confirmed that pure crystalline HAP was formed from all solvents used in this study. The solvents, however, played a critical role in tailoring the morphology of the HAP precipitates. Regular shape particles were formed from solutions of low dielectric constant such as ethanol and THF and irregular crystals were formed from a solvent with a higher dielectric constant such as water. The trend observed in the TEM images under the various conditions was consistent with the XRD results.

The rod-like shape HAP crystals with a diameter of ~ 6nm and length of 75 nm formed at room temperature in ethanol solvent using the rapid addition mode would be a promising starting point for the fabrication of in situ polymer-HAP composites for biomedical applications.


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Hadeel Alobeedallah (a), Jeffrey L. Ellis (b), Ramin Rohanizadeh (c), Hans Coster (a) and Fariba Dehghani (a)

(a) School of Chemical and Biomolecular Engineering, The University of Sydney, Sydney, 2006, Australia

(b) Department of Chemical and Biomolecualr Engineering, The Ohio State Univeristy, Coloumbus, OH, USA

(c) Faculty of Pharmacy, The University of Sydney, Sydney, 2006, Australia

Received 31 July 2010; Accepted 1 August 2010; Available online 1 March 2011
Table 1: Synthesis of nano-hydroxyapatite--A chronological development

Process                   Synthesis temperature   solvent

Spray drying              Heat treated at         water
Biomemetic                Heat treated at         Synthetic body
                          373[degrees]C           fluid (SBF)
hydroxyapatite/           Sintered at             n-butanol
  chitosan composite      400[degrees]C
Radio frequency           40 [+ or -]             water
  plasma spray            5[degrees] C and
                          sintered at
Sol gel                   85[degrees]C,           ethanol
                          sintering at
Wet Chemical              15-99[degrees]C         water
sucrose-templated         Heat treated at         water
  sol-gel                 850[degrees]C
Hydrolysis                75[degrees]C,           water
                          heat treated at
                          800[degrees] C
Citric acid sol-gel       70[degrees]C,           water
  combustion              HAP obtained at
                          75 0[degrees]C
Sol gel                   40[degrees]C/           Ethanol + water
                          80[degrees]C no
Rapid formation of        Microwave heating       Solution of
  hydroxyapatite na                               [Na.sub.2]H
  nostrps via microwave                           P[O.aub.4]
  irradiation                                     (0.06 M)
                                                  and CTAB(0.1M)

Process                   Particle size/shape   Ref.

Spray drying              20 nm/ spherical      [18]

Biomemetic                50 nm                 [19]
hydroxyapatite/           30 nm in width and    [14]
  chitosan composite      50-60 nm in length
Radio frequency           15|im/ spherical      [20]
  plasma spray

Sol gel                   1.3nm-2246nm in       [16]

Wet Chemical              Not reported          [21]
sucrose-templated         30-50nm/ spherical    [22]
Hydrolysis                50-500 nm             [23]

Citric acid sol-gel       Spherical/            [24]
  combustion              3 [micro]m

Sol gel                   Spherical at          [17]
                          at 80[degrees]C
Rapid formation of
  hydroxyapatite na
  nostrps via microwave   10-55 nm              [25]

CTAB (Cetyltrimethylammonium Bromide), SBF (Synthetic Body Fluid)

Table 2: Experimental conditions for the HAP synthesis

Solvent            Temperature    Addition mode

Ethanol            20[degrees]C   40[degrees] C

Tetrahydrofuran    20[degrees]C   40[degrees] C
Water              20[degrees]C   40[degrees] C

Solvent                           Addition mode

Ethanol            70[degrees]C   Slow
Tetrahydrofuran    70[degrees]C   Slow
Water              70[degrees]C   Slow

Table 3: XRD characteristics peaks for the prepared samples

Sample      Solvent    Synthesis     ([theta]B)   ([theta]1)

Sample a    Ethanol        20           31.8         31.7
Sample b    Ethanol        20           31.9        31.85
Sample c    Ethanol        40           31.9        31.86
Sample d    Ethanol        70           31.8        31.74
Sample e      THF          20           31.8        31.77
Sample f     Water         20           31.8        31.73
Sample g     Water         40           31.9        31.85
Sample h     Water         70           31.9         31.3

Sample      ([theta]2)   B = [DELTA]   Intensity
                           [theta]        Lin

Sample a      32.13         0.43          248
Sample b      32.06         0.21          343
Sample c      32.21         0.35          228
Sample d      31.89         0.15          278
Sample e      31.88         0.11          290
Sample f      31.93         0.20          118
Sample g      31.97         0.12          253
Sample h      31.37         0.07          283

Table 4: Assignments of IR bands
of HAP

Associated      Bonds
FTIR wave

564             O-P-O bending
602             O-P-O bending
980             P-O asymmetric
1036            P-O asymmetric
1091            P-O asymmetric
624             O-H in-plane
3450            O-H stretching

Table 5: Prepared HAP Particles Characteristics

Sample     Solvent    Synthesis     Addition
                     temperature      mode

Sample a   Ethanol        20          slow

Sample b   Ethanol        20         rapid

Sample c   Ethanol        40          slow

Sample d   Ethanol        70          slow

Sample e     THF          20          slow

Sample f    Water         20          slow

Sample g    Water         40          slow

Sample h    Water         70          slow

Sample          Particles        Thickness     Length
                  shape            (nm)         (nm)

Sample a    rod like crystals        7           66

Sample b    rod like crystals        6           75

Sample c   spherical crystals       105          --

Sample d    rod like crystals       14          103

Sample e   spherical crystals       93           --

Sample f   irregular spherical   irregular       --
Sample g     irregular rods         --       irregular
Sample h        irregular        irregular   irregular
           (spherical & rods)    (70-240)     (32-119)
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