The fields of application of calcium phosphates (CaPs) in orthopaedic surgery comply ceramic implants, mouldable cements or granulates as well as bioactive coatings on metallic implant materials [1–3]. The mechanical stability of CaPs is relatively low, hence the use of these is mainly focused on non-load-bearing applications in the cranio- and maxillofacial region as well as in the orbital region  and on the filling of bone defects in regenerative periodontal surgery . A promising material class of CaPs, which combines the advantages of free mouldability and mechanical stability, are the so-called self-setting bone cements (CPC) [6–8]. Although various mixtures of CaPs can serve as basic materials, there are in principle only two cement types as products of the setting reaction: At neutral or basic pH the CaP cement sets to nanocrystalline hydroxyapatite (HA), while a pH below 4.2 will lead to the formation of brushite .
One frequently used cement systems is based on mixtures of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA), which reacts – depending on the stoichiometric ratio of the reactants – to HA (Ca5(PO4)3OH) or Ca-deficient HA (Ca9(PO4)5HPO4OH, CDHA) with Ca/P ratios ranging from 1.5 to 1.67 [10–16]. The setting process of this cement consists of the partial dissolution of the educts and the precipitation of the less soluble product HA. Both processes take place simultaneously until the whole starting material is consumed or the precipitated material inhibits the diffusion and hence disrupts the setting reaction. The optimal stoichiometry with respect to HA precipitation is only achieved for this cement system, when the reactants dissolve congruently at the same rate. Since the dissolution rates of TTCP and DCPA are highly different, both components have to be provided in suitable and well-defined particle sizes. The grain size of TTCP granules should be in the range of 10–20 µm, while the DCPA has to be much finer with a grain size <2 µm [17, 18]. To obtain such fine sized particles, long and intensive grinding procedures are required, which may take up to 24 h of treatment in liquid phase [19, 20]. Besides the long processing time this procedure has also the disadvantage that the cement powder may be contaminated with wear particles from the grinding tools. In addition, the subsequent drying procedure is problematic and often connected with reduced reactivity of the ground powder as a result of contamination of the particle surface with solvent residues.
The aim of this study was to obtain secondary calcium phosphate powders (DCPA) with a high reactivity immediately from a precipitation reaction. As precipitation from homogenous aqueous solution usually results in much bigger grain sizes of the product, an alternative approach based on water/oil emulsions was applied, which had already been successfully tested for the fabrication of brushite granulates with optimized morphology for bone filling applications . In principle, the spatial limitation of the water droplets in the emulsions was supposed to inhibit crystal growth and should therefore lead to the formation of smaller particles. The obtained particle batches were characterized regarding particle size distribution, phase composition and grain morphology. After mixing the differently produced DCPAs with equimolar amounts of TTCP, the resulting cements were characterized regarding setting time and pH value of the cement paste as well as compressive strength and phase composition of the completely set cements. The results were compared with a reference material obtained from powders, which were prepared according to the conventional grinding method.
Emulsion synthesis of dicalcium phosphate (DCPA)
It was found that the application of the dispersing device led to the formation of cloudy OW emulsions, when the oil content was 70 or 80%, but yellowish and turbid WO emulsions of low viscosity were obtained for an oil content of 90% in which CaP crystal formation could be shown inside the water droplets (Figure 1A). These particles appeared to be agglomerates (>20 µm) of fine-sized crystals with a size in the sub-µm range (Figure 1B). This was confirmed by particle size measurements demonstrating medium sizes d50 to be in the range of 6–10 µm, while the primary crystal size (calculated from peak widths of XRD data) was found to be approximately 30–90 nm (Figure 2).
X-ray diffraction patterns of the obtained crystals showed the presence of monetite as the predominant phase for all CaP powders prepared by emulsion synthesis, whereas a minor phase of brushite could be observed (Figure 3).
The obtained monetite powders were mixed with tetracalcium phosphate (prepared by sintering) in an equimolar ratio. These cement powders were reacted with a 2.5% sodium phosphate solution and set for 24 h. The properties of the set cements are summarized in Table 1. A reference cement was made by using 24 h ground commercial DCPA which showed a setting time of 24 min and a compressive strength of 5.4 MPa. The setting times of cements prepared with DCPA from emulsion synthesis were much shorter and in a range of 7–15 min; their strength was increased to 12–17 MPa, while the cement density was not influenced.
Cement setting occurred at a slightly alkaline pH value with a continuous pH increase for the reference cement to a final value of approx. 11.2, while the use of DCPA prepared by emulsion synthesis led to a pH plateau of 9.5 after 2 h (Figure 4). X-ray diffraction analysis of the set cements (Figure 5) revealed the formation of nanocrystalline hydroxyapatite for all cements with only minor amounts of unreacted monetite or tetracalcium phosphate as further phases. A Rietveld refinement analysis of the XRD data (Table 2) indicated a degree of reaction to hydroxyapatite of approximately 80% for the reference cement after 24 h setting, while those cements with DCPA made by emulsion synthesis showed a higher HA content of >90%. Although the average HA crystal size was quite similar for set cements (approx. 12–14 nm), the HA size in the crystallographic 002 direction was significantly higher (from about 60% to 170%, see Table 2), which indicates preferred crystal growth into this direction.
Calcium phosphate cements are mixtures of calcium orthophosphate salts which set to form a matrix of either hydroxyapatite or brushite after mixing with an aqueous phase . The particle sizes in CPC are much smaller than those in civil engineering Portland cements  to ensure a high reactivity and fast setting reaction. Processing of cement components involves sintering of CaP mixtures to obtain high temperature phases (e.g., TTCP, TCP) as well as prolonged grinding to adjust the average particle size to the µm or submicron level. Nanosized CaP particles can be obtained without grinding by wet chemical precipitation methods as described in literature [24, 25], however this is only possible for CaPs like tricalcium phosphate (whitlockite) or hydroxyapatite with a small rate of crystal growth (2.7×10-7 mol Ca5(PO4)3OH min-1 m-2) . Secondary phosphates (brushite, monetite) have a by several orders of magnitude faster rate of crystal growth [e.g. 3.32×10-4 mol DCPD min-1 m-2 ], such that DCPD or DCPA crystals immediately grow to a size >1 µm.
The current study used a different approach to obtain fine sized CaP particles for cement synthesis. According to a study by  the aim was to use water in oil emulsions to limit the space for particle precipitation and following crystal growth, such that the preparation of nanoparticles from the secondary CaP phases brushite and monetite will be possible without further grinding procedures. Indeed, the primary crystal size of the precipitates obtained in our study was in the range of 30–90 nm, whereas the crystals formed larger agglomerates in the µm range (Figure 2). While the type of surfactant seemed to have an influence on the crystal size with Tween80 resulting in significant larger crystals compared to Tween20 or Brij35, no systematic effects of the surfactant concentration on the crystal size or the phase composition were found (Figures 2 and 3). Surprisingly, the precipitated crystals were predominantly composed of the anhydride monetite, although the reaction was carried out at room temperature similar to the reaction conditions of . This may be due to the shorter aging time of the emulsions in our study (1 d) compared to the 8 d applied there before demulsification. Aging of the emulsions in our study also resulted in pure brushite after 3 d (data not shown) indicating that the initially formed monetite was successively transformed to brushite by stepwise recrystallization and incorporation of water during prolonged aging.
All DCPA powders produced by emulsion synthesis were highly reactive when combined with an equimolar amount of basic tetracalcium phosphate and formed nanocrystalline hydroxyapatite in a cement setting reaction (Figure 5). The higher reactivity compared to ground DCPA could be concluded from a strong decrease of the setting time from 24 min to 7–15 min (Table 1). Cement setting occurs by simultaneous dissolution of both cement components followed by precipitation of HA from the supersaturated liquid cement phase. Since TTCP is a strong basic phosphate [pH in water approx. 12.5–13 ] and DCPA is slightly acidic (pH in water approx. 6–7), the pH of the cement paste during setting is an indicator for congruent dissolution of both cement reactants. The reference cement with DCPA being ground for 24 h showed a continuous increase to a pH of approx. 11.2 after 5 h, which indicates that the DCPA component has a lower rate of dissolution than the TTCP phase. In contrast, the pH evolution in cements containing DCPA made by emulsion synthesis reached a plateau around 9.5, at which both components dissolve at the same rate. It is assumed that at this stage the precipitation conditions of hydroxyapatite are optimal according to the overall setting reaction:
This high reactivity can be confirmed by the quantitative phase compositions of the cements after 24 h, which showed <10% TTCP and <1% DCPA for these cements compared to 18% TTCP (2% DCPA) for the reference material. Interestingly, the different reaction conditions in the liquid cement phase have also affected the crystal growth behaviour of HA as setting product. Although the average crystal size was found to be similar to that of the reference cement, a preferred growth in (002) direction (based on the peak width at 2Θ=25.9º) was observed.
The mechanical performance of such calcium phosphate cements is of interest for an application in low load-bearing areas. Mechanical strength of CPC is a result of a three-dimensional entanglement of cement crystals and is influenced by several factors such as the type of setting product (hydroxyapatite or brushite), the degree of conversion  or the porosity of the cement matrix . The latter is formed by the aqueous cement phase which is the reaction medium for dissolving cement components and precipitating the setting product. In contrast to brushite cements, water used for cement paste mixing is only marginally taking part in the setting reaction of HA cements and is hence mainly responsible for the porosity of the set cement matrix. Although the porosity of all cements was very similar since the same amount of water was used for paste mixing (Table 1), compressive strengths were found to be much higher for the cements containing DCPA from emulsion synthesis (12–17 MPa) compared to the reference cement with ground DCPA (5.4 MPa). The reason is likely the higher degree of conversion as described above and probably the higher aspect ratio of the precipitated HA crystals, which are longer in (002) direction and hence may lead to a better entanglement of the crystals in the set cement matrix.
However, regardless of the most promising results, it should be stated that rapeseed oil and the surfactants used may leave undesired organic residues in the cements, which could not be detected with the applied measuring techniques. Future studies should also include a thorough investigation of the purity of the final product, using suitable methods like MALDI-MS.
This study showed the successful synthesis of secondary CaP particles in an emulsion technique for the use in self setting calcium phosphate cements. The formulation of an HA cement from the novel DCPA powders and TTCP led to significantly improved compressive strengths, an increased conversion rate of the starting materials to HA, and reduced setting times in comparison to HA cements, which were produced from monetite particles ground to 1 µm according to the classical method. Hence the production of calcium hydrogen phosphate with an emulsion technique can be regarded as a promising alternative to the traditional powder processing by grinding methods. The greatest advantages lie in the high phase purity of the resulting starting materials and the considerably improved properties of the set cements.
Materials and methods
Emulsion synthesis of CaHPO4 powders
The oil/water emulsion system for the alternative preparation of the DCPA powders consisted of deionized water and rapeseed oil with an oil-to-water volume ratio of 9:1. Two aqueous mother solutions of CaNO3·4H2O and NH4H2PO4, each with a total volume of 30 ml, a concentration of 1.25 mol/l and with the addition of 5–10% surfactant (Brij35, Tween20 or Tween80, all Merck, Germany), were prepared. The emulsification was carried out using a high-performance dispersing device (T50 Basic Ultra Turrax, IKA, Staufen, Germany) at 10 000 rpm. The aqueous mother solutions provided with the surfactant were given into polyethylene flasks, covered with a layer of the given amount of oil and dispersed for 5 min. The resulting partial emulsions were subsequently mixed, dispersed for another 5 min and finally aged for 1 d. The aged emulsions were then demulsified by addition of 50 Vol.% acetone and centrifugation at 4500 rpm for 15 min. Subsequently the precipitate was dispersed in acetone and again centrifuged for 15 min. This rinsing procedure was performed three times and the cleaned powder was dried at 40°C.
The structures of the different emulsions were observed by transmission microscopy with an Axio Imager (Zeiss, Oberkochen, Germany). Morphological studies of the precipitated DCPA powders were carried out using a scanning electron microscope DSM 940 (Zeiss, Oberkochen, Germany). Prior to measurement the particle surfaces were coated with gold to avoid surface charge build-ups. The particle size distribution was determined using a laser particle size analyzer (L300, Horiba, Kyoto, Japan) with hundred milligrams of the powder particles suspended in 50 ml isopropanol and dispersed by applying ultrasound for 15 min.
Cement preparation and testing
The cement used in this study was composed of an equimolar mixture of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA). TTCP was prepared by sintering an equimolar mixture of DCPA (Mallinckrodt-Baker No. 1430-07, Griesheim, Germany) and calcium carbonate (Merck, Darmstadt, Germany) at 1500°C for 6 h followed by quenching in air. The sintered cake was crushed with pestle and mortar and sieved to a size <160 µm. Subsequently the material was milled in agate jars in dry state to a medium particle size of 10–15 µm, which was determined by a laser particle sizer Horiba LA-300 (Kyoto, Japan). For the reference measurements the second cement component DCPA was prepared according to the common method by ball-milling in 96% ethanol for 24 h and afterwards dried in vacuum at 60°C.
For the preparation of the cement pastes 6.68 g TTCP and 2.7 g of the differently produced DCPA powders were mixed in a coffee grinder for 45 s. Afterwards an aqueous solution of 0.25 mol/l Na2HPO4 and 0.5 mol/l trisodium citrate was added to obtain a cement paste with a powder-to-liquid ratio of 2.7 g/ml. The development of the pH value of the cement pastes during the setting process was measured for up to 5 h in intervals of 5 min using an insertion pH electrode. Uncompacted cuboid samples were obtained by manually filling the cement paste into flat silicone moulds (6 mm×6 mm×12 mm). The compressive strengths of wet samples (n≥6) after 24 h hardening at 37°C were measured along the long axis using a static mechanical testing machine ProLine Z010 (Zwick/Roell, Ulm, Germany) with a 5 kN load cell and at a crosshead speed of 1 mm/min. The initial setting times of the cements were measured in a humidity chamber at 37°C and >90% humidity using the Gilmore needle test with a needle of 113.4 g and 2.117 mm diameter according to ASTM standard . Phase analysis of the set cements was performed by X-ray diffraction with a D5005 diffractometer (Siemens, Karlsruhe, Germany). Data were collected in a 2θ range from 20–40° with a step size of 0.02° and a normalized count time of 1 s/step. The phase composition was checked by means of JCPDS reference patterns for TTCP (PDF Ref. 25-1137), HA (PDF Ref. 09-0432) and DCPA (PDF Ref. 09-0080). Quantitative phase compositions of the materials were calculated by means of total Rietveld refinement analysis with the TOPAS software (Bruker AXS, Karlsruhe, Germany) with reference database structures of TTCP, HA and DCPA. The same software was used to calculate – based on the application of the Scherrer formula – the average crystal size by refinement of the whole diffraction pattern as well as the crystal size in 002 direction by restricting the refinement to the respective peak.
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