Synthesis Method of Hydroxyapatite

Author: Ceram Research Ltd

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Introduction
Due to the ageing UK population, increased dynamism of people’s lives and growing life expectancy there is an increasing clinical demand for bone replacement and repair. The main mineral component of bone tissue is a nonstoichiometric carbonated multi-substituted apatite with calcium to phosphorus ratio (Ca:P) between 1.37 and 1.87 [1]. Synthetic hydroxyapatite (Ca10(PO4)6(OH)2 - HA) is a popular bone replacement material because it has a similar crystal structure (Ca:P ratio fixed at 1.67) to native bone apatite. This resemblance is the origin of the excellent compatibility that HA exhibits with hard tissue and its natural bioactive behaviour; enabling it to be incorporated into the body via the same processes active in the remodelling of healthy bone. Currently, HA is used in a number of different applications as clinical solutions to bone defects caused by trauma or disease. These include:     Inert implant coatings (to strengthen bone-to-implant bonding) Porous granules as void or defect fillers Synthetic bone grafts Hard tissue scaffolds

In addition, HA has potential uses in other areas of medicine, for example as an inorganic drug delivery entity. To suit its numerous applications, material properties such as bioactivity and mechanical strength need to be tailored accordingly. Consequently, much research has been devoted to the development of synthesis methods that enable the control of chemical (e.g. crystallinity, Ca:P) and physical (e.g. porosity, particle size) powder characteristics. This paper presents a comparison and overview of the common preparation methodologies for HA.

Synthesis Methods
Due to the existence of a myriad number of phosphate compounds the calcium phosphate system is highly complex. This is further complicated by the sensitive stability of phosphates to minor changes in: composition, pH, and reaction conditions (e.g. temperature). It is important to note that the purity and particle characteristics of the final synthesised powder can affect the bioactivity, mechanical and biological dissolution properties. These characteristics ultimately determine the medical application of the material, thus making it imperative to develop a synthesis method that enables the control of: crystal morphology, chemical composition, crystallinity, particle size distribution, and agglomeration. A variety of synthesis techniques have been published for HA. Precipitation:It is maintained in the literature that conventional wet chemical precipitation methods are one of the most widespread approaches due to their simplicity, ready availability and use of relatively inexpensive raw materials. Combined with the use of low reaction temperatures, the result is minimal operating costs. Additionally, this process is attractive to manufacturing applications due to its scalability. However, due to the simultaneous occurrence of nucleation, crystal growth, coarsening and/or agglomeration, precipitation of HA cannot be viewed as
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trivial. Reactions require fine-tuning to optimise morphology and minimise crystal growth. Furthermore, a number of surfactants (e.g. PVA) or dispersants (e.g. ammonium polyacrylate, ethanolamine) have been investigated with the aim of reducing particle agglomeration, which occurs due to the high particle energy associated with small crystal sizes. In addition to tweaking reaction conditions, a subsequent time-consuming high temperature heat treatment may be needed to maximise the percentage of crystalline phase in the precipitated calcium phosphate. Supersaturation is the key to any precipitation process. A solution is defined as supersaturated when it contains more solute than should be present at equilibrium. Nucleation and crystal growth occur once the solution is supersaturated; this occurs when the phosphate solution is titrated into the calcium solution forming a suspension of precipitated particles. Final composition is dependent on the solution pH, concentration, and temperature - thus these reaction parameters must be controlled in Calcium Phosphate Growth after SBF Immersion order to generate a homogeneous product. Post-formation precipitated powders are typically calcined at 400 - 600°C to refine the crystal structure. In some cases, fully crystallised HA is not formed until it has been sintered up to 1200°C. Hydro/solvo- thermal: Hydro- and solvo-thermal processing involves the use of a solvent (with precursor soluble ions), which is heated in a sealed vessel. In the case of hydrothermal synthesis the solvent used is water. The temperature of the solvent can be brought above boiling point as the autogenous pressure within the vessel exceeds the ambient pressure. The change in solvent and reactant properties (e.g. solubility) at these elevated temperature mean that experimental variables can be controlled to a higher degree. This makes the reaction more predictable as crystal nucleation, growth, and ageing can be regulated. These methods are environmentally significant as they can be performed at substantially lower temperatures to solid-state reactions. Furthermore, other low temperature methods such as wet chemical precipitation and sol-gel synthesis require post heat treatment to crystallise the HA; whereas crystalline HA can be produced in one step via hydro- and solvothermal synthesis. Yields approaching 100%, relatively low cost reagents and short reaction times have also been reported for these processes. However, in reality the scalability of these “batch” techniques are limited to the size of the reaction vessel. Furthermore, solvent and surfactant selection may require modification to optimise production, which may be a lengthy process as relatively few controllable methods are reported within the literature. Solid state: Despite being a traditional method there are relatively few reports of solid-state synthesis of HA. This procedure relies on the solid diffusion of ions amongst powder raw materials and thus requires relatively inefficient high temperature processing (< 1250°C) to initiate the reaction. Even though the technique is comparatively simple there are a number of processes involved. To ensure homogeneity and sufficiently small particle sizes, starting materials must firstly be ball milled for approximately 16 hours. Commonly calcium and phosphate sources are mixed with additives (e.g. silicon dioxide, alumina, and hydrofluoric acid), a binder (e.g. PVA) and an organic
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vehicle (e.g. acetone) to form a slurry before milling. The slurry must then be dried. Pellets can then be formed from the resulting powder using either hot or cold pressing at pressures of up to 135MPa. Finally, sintering is performed at up to 1250°C to crystallise the product. After this final stage Pramanik et al. [2] suggest recrushing the sintered samples followed by further compaction and sintering at 1250°C to refine the crystal structure, and reduce the grain (7-8µm) and crystal size (50-70nm). Despite reports of the production of single phase HA via this method it is highly likely that α-tricalcium phosphate (Ca3(PO4)2 – TCP) may be present in the final product due to the high temperatures required to initiate the reaction. The α-TCP phase may then be transformed to β-TCP during sintering; this is reported to occur at 1063°C [3]. If there is any incorporation of different calcium phosphate phases in the final product the dissolution and mechanical properties of the material will be altered, which adversely makes its performance unpredictable. Sol-gel: Recently, sol-gel techniques have attracted much attention due to the inherent associated advantages of this method; homogeneous molecular mixing, low processing temperatures (<400°C), and the ability to generate nano-sized particles. However, the energy saving gained from the low temperatures used is offset by the high cost of the reactants. In comparison to other low temperature methods, sol-gel techniques have very limited scalability due to the sensitivity of the process. The first stage of this method is to form a ‘sol’; a dispersion of solid particles, otherwise known as colloids, in a liquid. Precursor materials are mechanically mixed in a solvent at a pH that prevents precipitation. Typically, metal alkoxides (e.g. tetraethoxysilane to introduce silicon) and metal salts (e.g. calcium nitrate to add calcium; ammonium phosphate to add phosphorus) are used. Hydrolysis and polycondensation reactions occur to link these monomer units and form M-O-M bonds within the sol causing the viscosity to increase; this process is termed gelation. The result is a ‘gel’, which can be defined as a diphasic system consisting of a solid and interstitial liquid phase. It should be noted that forming a gel without flocculation can be difficult. The next step is to remove the liquid phase via a drying process; this is usually accompanied by a significant amount of shrinkage and densification. To avoid cracking in a target 3-D monolith structure it may be necessary to age the gel before drying. Alternatively, cracking can be accommodated if the goal is to create a fine sol-gel powder for further processing as a granulate or shaped product.Lastly, a material specific sintering protocol is employed; in practise this step can be timeconsuming. Self propagating combustion synthesis: Self propagating combustion synthesis (SPCS) has recently been proposed as a viable simple, quick energy saving synthesis option for HA [4]. Interestingly, Ceram investigated SPCS as a possible route to generating low particle size pigments for ceramic ink-jet printing applications during the 1990s. The success of SPCS is dependent on the intimate mixing of constituents in an aqueous medium: calcium and phosphate sources, a suitable fuel (e.g. urea, citric acid, tartaric acid, glycine, sucrose, or succinic acid), and an oxidising agent (e.g. nitric acid). The solution temperature is then increased causing a vigorous exothermic reaction to occur between the fuel and oxidiser; the gaseous products of this reaction spontaneously combust. This gives rise to a very high local temperature that causes the formation of a solid calcium phosphate powder. This process can be completed in less than 20 minutes. The heat energy evolved from the reaction is dependent on the fuel used as well as the ratio of fuel to oxidiser. These parameters can be varied, resulting in the
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formation of different calcium phosphate phases and/or particle morphologies. Furthermore, depending on the fuel used the product formed may be either crystalline or amorphous. Both require a calcination step; to remove organic residues and crystallise the phase formed, respectively. Crystalline HA has been synthesised via this method using tartaric and sucrose as fuels. However, little work has been carried out to determine the sensitivity of the reaction to changes in fuel to oxidiser ratio and the affect this has on the phase and morphology of the product formed. Without completing a more in depth investigation the viability of using this method on a large scale is uncertain. However, it is expected that it will be unfeasible due to (a) the potential for incomplete reaction resulting in mixed phase powders and (b) the uncontrollable reaction front developed during combustion of gaseous products [5]. Emulsion/Micro-emulsion: Emulsions are heterogeneous mixtures of at least one immiscible liquid dispersed in another in the form of droplets [6]. These systems are often described as either water-in-oil (W/O) or oil-in-water (O/W); the first phase stated is the dispersed one and generally O/W systems are used to make HA. Depending on the size of the aqueous drops, i.e. the size of the reaction domains, this technique can be referred to as emulsion or microemulsion. A reaction takes place when two different droplets containing the reactants collide with each other. Nano- and micro-sized particles of HA can be formed via microemulsion and emulsion, respectively. Furthermore, microemulsion techniques have been reported to reduce particle agglomeration of HA [7]. One specific application of this synthesis process is the formation of porous spherical HA granules for drug delivery [8]. Granules at 50 - 2000µm have been formed with porosity up to 58.5% and no substantial trace of impurities up to 1250°C via emulsion routes. However, the pores formed were poorly interconnected. This may hinder cell in growth, vascularisation, and diffusion of nutrients in vivo. Alternatively, HA particles produced via microemulsion routes with spherical morphology and small particle sizes (>22nm) can be used in plasma spraying due to their superior powder flow characteristics [7].

Conclusions
This overview demonstrates the vast variability of HA synthesised by some of the most popular methodologies. Distinctive differences in physical powder properties (e.g. particle size, agglomeration, and morphology) as well as chemical ones (e.g. Ca:P ratios and calcium phosphate phases) can be observed and indeed controlled. Different applications of HA require different physical and chemical properties thus the logical conclusion to draw from this report is that the optimum characteristics for each application should be decided before a synthesis technique is selected. This information can then be used to make an informed selection of the most appropriate technique to select and potentially develop. Furthermore, the ease of scalability and cost of each process are other factors to consider as these also widely fluctuate, especially if large quantities are required. Once a decision of synthesis route has been made, a full understanding of how process variables impact on HA powder product properties is required. Use of Factorial Experimental Design (FED) software can be very useful at this stage to determine which combination of variables have the greatest impact. FED studies can ultimately inform the process of creating robust Standard Operating Procedures (SOP).
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HA Synthesis Method

Method Description

Processing Reaction Time Temperature (</> 24hrs) (°C) Room temperature – 85

Particle Size (µm)

Raw Material Costs* 100% (£97.10)

Scalability

Comments  Most common method  Three step procedure; requires calcinations and sintering after processing  Difficulties with reproducibility due to lack of precise control  Homogeneous crystal shapes and sizes  Single step process to form crystallised HA  Cost dependent on particle morphology  Most traditional method  Involves long ball milling, drying, compression, and sintering steps  May need to repeat process steps to improve quality and reduce particle size  Slow and difficult to achieve a complete reaction; mixed phase product  Molecular mixing improves chemical homogeneity  Difficult to form single phase  Involves more than 3 processing steps  HA prepared exhibits inferior crystallinity and thermal stability  Reaction time <20mins  Uncontrollable high temperature reaction front may lead to mixed phases  Particle morphology dependent on fuel used  Porous particles can be formed  High temperature calcination and sintering steps required  Highly dependent on oil and surfactants used

Refs

Calcium and phosphate solutions combined under Wet-chemical controlled reaction parameters Precipitation resulting in nucleation and crystal growth Hydro- and Solvo-thermal Reaction takes place in aqueous solution in a closed system under conditions of high temperature and pressure HA formed via solid-state diffusion of ions from solid reactants. Requires thermal treatment to initiate reaction

>

> 0.1

High

[9 – 13]

<

150 – 400

> 0.05

89 – 175%

Low

[14 – 23]

Solid State Reactions

>

1050 - 1250

> 2.0

276%

Medium

[2, 3, 24]

Sol-gel

‘Sol’ solution is formed that evolves into a ‘gel’ system via hydrolysis and polycondensation Particles formed from aqueous solution via spontaneous combustion of a fuel and oxidiser at elevated temperatures Droplets of immiscible liquids react in a heterogeneous mixture

>

37 - 85

> 0.001

120 – 180%

Low

[25 – 28]

SPCS

<

170 – 500

> 0.45

118 – 136%

Expected to be low

[4, 5, 29]

Emulsion and Microemulsion

>

Room temperature – 50

> 1.0 (emulsion) > 0.005 (micro)

163%

Low – Medium

[6 – 8, 30]

*Based on 1kg of Calcium source, 500g of Phosphorus source and minimum order amounts of any additional reactants. Prices quoted from Sigma Aldrich website 07/2012.
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References
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[19] Guo, X.Y. and P. Xiao, Effects of solvents on properties of nanocrystalline hydroxyapatite produced from hydrothermal process. Journal of the European Ceramic Society, 2006. 26(15): p. 3383-3391. [20] Wang, Y.J., et al., Investigations on the formation mechanism of hydroxyapatite synthesized by the solvothermal method. Nanotechnology, 2006. 17(17): p. 4405-4412. [21] Zhang, S.Y., et al., Kinetic studies on the synthesis of hydroxyapatite nanowires by solvothermal methods. Australian Journal of Chemistry, 2007. 60(2): p. 99104. [22] Ma, M.G. and J.F. Zhu, Solvothermal Synthesis and Characterization of Hierarchically Nanostructured Hydroxyapatite Hollow Spheres. European Journal of Inorganic Chemistry, 2009(36): p. 5522-5526. [23] Yang, C., et al., Solvothermal synthesis and characterization of Ln (Eu(3+), Tb(3+)) doped hydroxyapatite. Journal of Colloid and Interface Science, 2008. 328(1): p. 203-210. [24] Rao, R.R. and T.S. Kannan, Synthesis and sintering of hydroxyapatite-zirconia composites. Materials Science & Engineering C-Biomimetic and Supramolecular Systems, 2002. 20(1-2): p. 187-193. [25] Liu, D.M., T. Troczynski, and W.J. Tseng, Water-based sol-gel synthesis of hydroxyapatite: process development. Biomaterials, 2001. 22(13): p. 1721-1730. [26] Bezzi, G., et al., A novel sol-gel technique for hydroxyapatite preparation. Materials Chemistry and Physics, 2003. 78(3): p. 816-824. [27] Weng, W.J., et al., The effect of citric acid addition on the formation of sol-gel derived hydroxyapatite. Materials Chemistry and Physics, 2002. 74(1): p. 92-97. [28] Agrawal, K., et al., Synthesis of HA by various sol-gel techniques and their comparison: a review. National Conference on Advancements and Futuristic Trends in Mechanical and Materials Engineering, 2011. [29] Sasikumar, S. and Vijayaraghavan, R., Solution combustion synthesis of bioceramic calcium phosphates by single and mixed fuels – a comparative study. Ceramics International, 2008. 34: p. 1373 – 1379 [30] Koumoulidis, G. C., et al., Preparation of hydroxyapatite via microemulsion route. J Colloid Interface Science, 2003. 259: p. 254 - 260

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About Ceram
Ceram is an independent expert in innovation, sustainability and quality assurance of materials. With a long history in the ceramics industry, Ceram has diversified into other materials and other markets including aerospace and defence, medical and healthcare, minerals, electronics and energy and environment. Partnership is central to how we do business; we work with our clients to understand their needs so that we can help them overcome materials challenges, develop new products, processes and technologies and gain real, tangible results. Headquartered in Staffordshire, UK, Ceram has approved laboratories around the world.

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