Effects of high energy dry milling on biphase calcium phosphates

Effects of high energy dry milling on biphase calcium phosphates R. Ilieva1, E. Dyulgerova2, O. Petrov*3, R. Aleksandrova4 and R. Titorenkova3 The effect of high energy dry milling on the structural and crystalline state of sintered biphase calcium phosphates was studied. After various periods of grinding, the initial biphase calcium phosphate material alters its crystalline structure and phase composition. The phase transformations achieved during milling were recorded by powder X-ray diffraction, scanning electron microscopy, infrared spectroscopy and chemical analysis. X-ray diffraction analysis of samples milled for 20 h showed that the initial composition of the biphase ceramics changed and part of btertiary calcium phosphate was nanocrystalline and partially in amorphous state. Hydroxylapatite fully transformed into nanocrystalline phase. Cytotoxicity tests of samples milled for 20 h clearly present cellular viability with pronounced biological activity. Keywords: Biphase calcium phosphate, Sintering, Mechanochemical structural phase transformations, Cellular viability

Introduction Recently, there is a constantly growing interest in sintered biphase calcium phosphates (BCPs) hydroxylapatite (HAP) and b-tertiary calcium phosphate (b-TCP) used as scaffold materials, with chemical composition close to the mineral part of the bone (with molar ratio of Ca/P near 1?67) and solubility favourable for the biomineralisation around the bone implant materials.1 It is known that BCP materials are more effective in bone repair and regeneration than monophase HAP or b-TCP ones, and have a controllable degradation rate to a certain degree.2–7 Composites of b-TCP and HAP biphase phosphates combine the excellent bioactivity of the two phases: the good osteoconductivity of HAP and the high properties of resorbability (bioresorbability) of b-TCP.8,9 Thus, these different properties of the two phases give impetus for study of such biphase type of Ca phosphates. Understanding the synthetic characteristics and structural chemistry of calcium phosphate materials is a key into their improved medical application, but their chemistry is complex, offering diverse structure with intrinsic compositional variation.10 In this point of view, there are a lot of possibilities to approve the quality of advanced biomaterials. In order to apply appropriate BCPs to meet specific biological need, it is crucial to control BCPs with various ratios of HAP/TCP. Furthermore, recently, nanobioceramics have attracted attention for their effective bioactive properties.11–16 1

Institute of General and Inorganic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bontchev Str., Bl. 11, Sofia 1113, Bulgaria Dental Medicine Faculty, University of Medicine, 1 G. Sofiiski Str., Sofia 1431, Bulgaria 3 Institute of Mineralogy and Crystallography, Bulgarian Academy of Sciences, Acad. G. Bontchev Str., Bl. 107, Sofia 1113, Bulgaria 4 Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Science, Acad. G Bontchev Str., Bl. 25, Sofia 1113, Bulgaria 2

*Corresponding author, email [email protected]

ß 2012 Institute of Materials, Minerals and Mining Published by Maney on behalf of the Institute Received 17 August 2011; accepted 2 September 2012 DOI 10.1179/1743676112Y.0000000063

Advances in Applied Ceramics

Advances in Applied Ceramics aac995.3d 20/9/12 20:53:13 The Charlesworth Group, Wakefield +44(0)1924 369598 -

Many studies have shown that bone forming cells for specific application are interacting with nanoscale surfaces of biomaterials, which is critical to keep the body from rejecting artificial parts17,18 and promote the adhesion, proliferation and differentiation of osteoblasts.19,20 Nevertheless, the relationship between the characteristics of crystals and their biological activity is still unknown. Despite difficulty in accurately characterising the composition, the structural details and the properties of calcium phosphate nanocrystals, the investigations in this direction make intense progress. It is essential to develop the use of apatite nanocrystals in biomaterials and to reach a better understanding of the role and behaviour of the bone mineral.21 Some physicochemical parameters such as surface topography, particle size and tridimensional structure of biomaterials are factors in bone regeneration and repair processes. Similar situation exists in the fields of nanocrystalline apatite considered to play a major role in the biologically active materials,21,22 but they remain rather poorly defined and characterised. The poor knowledge and characterisation of calcium phosphate nanocrystals may result in inconsistencies and disagreement in experimental results. Mechanical milling of solid state material is a method that takes advantage of the perturbation of surface bonded species by pressure to enhance their thermodynamic and kinetic reactivity. In addition, it is one of the promising techniques to receive nanosized material. Different morphologies, stoichiometries and crystallinities can be obtained by mechanical treatment processes depending upon the technique and the materials used.23–28 Dispersion in grinding leads to radical changes in microstructure. Not only does grinding increase the surface area of particles, it also changes the state of the surface layers. In addition, defects accumulated in the volume and different reaction centres are formed at the rupture zones.29

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Effects of high energy dry milling on BCPs

It is well known that one of the most interesting features of mechanical milling is the ability to produce nanosized powders. Deformation strain during milling depends generally on the mechanical treatment conditions and the nature of treated materials. As a result of increasing the milling time, the crystallite size decreases until a steady state particles size is approached. To the best of our knowledge, at present, there is distinct lack of studies in the high energy dry grinding of BCPs and the data considering that the results of structural changing and phase transformations of this material are missing. From such point of view, it must be mentioned that single phase b-TCP subjected to high energy grinding for 20 h gets fully amorphous30 and single phase HAP at the same conditions is transformed into nanocrystals. Up to now, there are still no data about phase transformations of BCPs during milling especially for transformation in nanostate of both HAP and b-TCP synthesised as new bone implant materials with complex 3B activity (bioactivity, biocompatibility and bioresorbability). In this context, it is of growing interest to study such processes in order to obtain biomaterials with new complex properties. The present work is based on a promising approach by use of dry milling process to prepare BCP nanomaterials with different morphologies and functionalities. The aims of the present study are to make attempt to evaluate the effects of high energy dry grinding of BCPs for different lag times until obtaining nanometric crystal size materials. For this purpose, BCP sintered samples are synthesised and characterised. Subsequently, they are subjected to high energy dry milling. The obtained materials are then studied by chemical, morphological and structural analyses.

Experimental Materials The starting material used in this study was poor crystalline apatite or non-stoichiometric apatite, synthesised by double decomposition of Ca(NO3)2.4H2O (1M in deionised water) and (NH4)2HPO4 (0?6M in deionised water) (Merck) to resave solutions with molar ratio Ca/P 51?67. The phosphate solution was dropwise poured with a rate of 4 mL min21 into the solution of Ca (NO3)2 at constant stirring and continuing keeping pH about 10?5–11 (adjusted by addition of NH4OH) at room temperature. The mixture was maintained in solution for 24 h at room temperature for aging, ensuring that the reagents were fully reacted and precipitated. The chemical reactions may be presented according to Ca(NO3 )2 z(NH4 )2 HPO4 ? Ca10{x (HPO4 )x (PO4 )6{x (OH)2{x zNH4 NO3 zH2 O (1) At the end of the maturation period, the precipitate was centrifuged, filtered and washed with deionised water. The resulting material was dried at 100uC for 10 h to receive poorly crystalline apatite and was denoted as starting material (A0). Afterwards, this sample was sintered stepwise from 800 to 1100uC for 1 h at heating rate 5uC min21, and the resulting samples were denoted as A for 800uC and A1 for 1100uC respectively. Depending on the degree of Ca deficiency x, the molar composition of the resulting biphase powder mixture of

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the calcinations .750uC can vary according to the following equation31,32 Ca10{x (HPO4 )x (PO4 )6{x (OH)2{x ? 3xCa3 (PO4 )2 z(1{x)Ca10 (PO4 )6 (OH)2 zH2 O

(2)

For x50, single phase HAP is formed, while for x51, TCP can be obtained and equal molar powders of both phases crystallise at x50?25. For these reasons, we characterised the processes in a step-like manner to follow calcination in the interval 800–1100uC. Subsequently, the sintered sample A1 (at 1100uC) was dry milled in agate planetary ball mill (‘pulverisette’ 6 Fritsch) at 600 rev min21, with agate bolls (10 mm diameter) in standard mass of sample ,30 g. The milling process was carried out for periods of times of 5, 10 and 20 h to obtain nanocrystalline sizes of the material. The mechanochemical treatment (high energy milling) increases the material specific surface area and changes the material structure, generating dislocation and point defects, which favour microarea reactivity. A small quantity of powder was taken from the mill chamber during different periods of time to be analytically investigated. The obtained milled samples are denoted as A1–B5, A1–B10 and A1–B20. This nomenclature is given in Table 1.

Methods The following methods were employed to study the phase transformation and physicochemical characteristics of all studied samples. The crystalline phases in all samples were determined by powder X-ray diffraction (XRD) with a Bruker D8 diffractometer, operating at 40 kV and 40 mA with Cu Ka radiation. This method was used to identify the crystalline phases present in the milling materials. Scans were performed over the range of 2h 10–80u (step size, 0?01; counting time, 1 s). The infrared (IR) spectra were collected by a Tensor 37 spectrometer (Bruker) and collected with a 4 cm21 resolution after averaging 72 scans on standard KBr pellets in the spectral region of 400–4000 cm21 at room temperature. The powders were mixed with IR quality KBr at a mass ratio of ,1 : 400, and the mixture was pressed into pellet. The adsorption spectra were acquired over the range of 400–4000 cm21 with a resolution of 2 cm21. Each spectrum was scanned 32 times to increase the signal/noise ratio. The estimated standard deviation of the wavelength was 4 cm21. Microstructure and morphological characterisation of the milled materials was studied on a JEOL JSM-5510 at a beam current of 20 mA and an accelerating voltage of 20 kV. Table 1 Nomenclature of studied samples Sample

Description

A0 A1 A1–B5

Dried materials at 100uC for 10 h Dried materials, sintered at 1100uC for 1 h Sintered at 1100uC for 1 h and mechanochemically treated for 5 h Sintered at 1100uC for 1 h and mechanochemically treated for 10 h Sintered at 1100uC for 1 h and mechanochemically treated for 20 h

A1–B10 A1–B20

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O–H stretching in HAP

A1_1100kontr

3572

A1_5h

3572

A1_10h

3571

A1_1100M20h

3572


CO3

n3 PO4

1414w

1121sh 1091 1118sh 1089 1120sh 1091 1090

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n4 PO4

1050

964

945

632

603

1051

945

631

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1052

971 962 965

944

632

603

1050

962sh

944sh

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OH libration in HAP

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n2 PO4 571 551sh 572 556 544sh 572 550sh 572

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amorphous b-TCP phase differing from fully amorphous monophase TCP in analogical experimental conditions.34 This finding would be appropriate for production of biphase nanocrystalline calcium phosphates with another third part of amorphous phase. These results support the hypotheses that they are relevant to positive biological responses as a consequence of dissolution processes and effects of higher local concentration of calcium and phosphate ions. Furthermore, in the microenvironment, elevated ion concentrations may define kinetics of bone-like apatite formations in bone/implant interface and bone implant material integrations.

Conclusions Samples of BCP sintered at 1100uC and high energy grinded for 20 h are transforming into nanosize crystalline form. The sizes are ,36 nm for HAP and ,33 nm for b-TCP. The ground materials are present in the form of nanoscale particles as nanocrystals and agglomerates of few micrometres with nanostructured surfaces seen in SEM. It was demonstrated that high energy grinding of BCPs is an easy method to obtain nanocrystallinity and agglomerates of biphase materials. The bioactivity of the obtained materials was demonstrated by studying the viability and proliferation of (Lep) cells. It was demonstrated that the high energy ground BCP materials have a higher bioactivity. The cell survival rate in the tests was as in the control. It is important to stress that in the BCPs b-TCP gets partially amorphous after 20 h of milling differing from the pure monophase of b-TCP, which becomes fully amorphous. There is a real expectation that these materials have a good potential in the field of functional advanced biomaterials.

Acknowledgement The authors thank the National Science Fund of Bulgaria (grant nos. DTK 02-70/2009 and DCAP-02/2/ 2009) for the financial support.

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