electron diffraction and high resolution … · 2 interdepartmental center of electron microscopy,...

European Cells and M aterials Vol. 1. 2001 (pages 27-42) DOI: 10.22203/eCM.v001a04 ISSN 1473-2262

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ELECTRON DIFFRACTION AND HIGH RESOLUTION TRANSMISSION ELECTRONMICROSCOPY IN THE CHARACTERIZATION OF CALCIUM PHOSPHATE

PRECIPITATION FROM AQUEOUS SOLUTIONS UNDERBIOMINERALIZATION CONDITIONS

E. I. Suvorova1 and P. A. Buffat2*1 Institute of Crystallography , Russian Academy of Science, Moscow, Russia

2 Interdepartmental Center of Electron Microscopy, Federal Institute of Technology EPFL, Lausanne, Switzerland

Abstract

Calcium phosphate precipitation obtained fromaqueous solutions at room and body temperatures and pH5.5-7.5 were investigated by high-resolution transmissionelectron microscopy (HRTEM), transmission electrondiffraction, scanning electron microscopy (SEM) and X-ray diffraction (XRD). Supersaturated solutions ofcalcium phosphates were prepared by different methodsof mixing of the stock solutions: diffusion-controlledmixing in space, convection-controlled mixing on earthand forced mixing on earth and with typicalphysiological parameters (pH and temperature).Concentrations of the stock solutions, rate of solutionmixing and duration of precipitation influence verystrongly the chemical composition of the precipitation,the phase composition of individual crystals, their sizes,morphology and structure. Microdiffraction and HRTEMtechniques showed an incontestable advantage on otherones like SEM and XRD to investigate small particlesand mixtures of calcium phosphates (hydroxyapatite andoctacalcium phosphate) with different proportions.

Key Words: Electron diffraction, high-resolutiontransmission electron microscopy, hydroxyapatite,octacalcium phosphate, precipitation, morphology, size,structure, microgravity

*Address for correspondencePhilippe A. BuffatEPFL, CIME, MXCCH-1015 Lausanne, Switzerland

Telephone number: +41 (0) 21 693 2983FAX number: +41 (0) 21 693 4401

E-mail: [email protected]://cimewww.epfl.ch

Introduction

Phase composition of calcium phosphate precipi-tation, morphology, sizes and structure of the individualparticles obtained from aqueous solutions are correlatedwith growth conditions. The knowledge of thiscorrelation is strongly necessary for a direct and rationalsynthesis of bioimplants. Also such calcium phosphatesas hydroxyapatite particles can be carriers of definiteadsorbed drugs injected into the body. Sizes, morphol-ogy and structure of the particles, which constitute thesebiomaterials, determine their chemical reactivity andbiomedical properties.

Calcium phosphate formation in aqueous solutionsunder biomineralization conditions can be served asimplified model of the bone tissue formation anddestruction on chemical and physical level. Therefore,the choice of the right methods and tools for determiningthe state of matter in the initial stage of precipitation is ofgreat practical consequence.

The present work stress out the limits of X-raydiffraction for calcium phosphates characterization,owing to the fact that the complex structure of OCP andHAP behave numerous lattice spacings that are veryclose, and the need to use a combination of electronmicroscopy tools in the direct space, as high resolutiontransmission electron microscopy HRTEM, and in thereciprocal spaces as microdiffraction

Several calcium phosphate modifications hexagonalhydroxyapatite (HAP), Ca10(OH)2 (PO4)6 (Kay andYoung, 1964), triclinic octacalcium phosphate (OCP),Ca8H2(PO4)6⋅5H2O (Brown et al, 1962), monoclinicdicalcium phosphate dihydrate or brushite (DCPD),CaHPO4 .2H2O, and triclinic dicalcium phosphate ormonetite (DCP), CaHPO4 can precipitate from aqueoussolutions. Among them only two general products, HAPand OCP, should be considered as materials that areprecipitating under biomineralization conditions. Suchconditions include diluted solutions with a typicalphysiological ionic strength (0.15 mol/l) at pH 5.5-7.5

E. I. Suvorova & P. A. Buffat Ca phosphates under biomineralization conditions

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and temperature ≤ 40°C. DCP and DCPD are acid saltsin comparison with the HAP and OCP phases and are theleast soluble for pH ≤ 4.2 according to the solubilitydiagrams (Fernandez et al., 1999a). In addition, DCP isusually obtained by heating DCPD at temperaturesbetween 120 and 170°C (Fernandez et al., 1999b).

This paper reports the results of examination ofcalcium phosphate specimens obtained from aqueoussolutions under different conditions of precipitation. Italso shows that only a combination of high-resolutiontransmission electron microscopy (HRTEM) with imagecalculation, microdiffraction and image processingprovides full information on the state of matter, localphase composition of crystals and atomic structure whensmall particles and small areas on large samples areconsidered.

Materials and methods

Supersaturated solutions of calcium phosphates wereprepared by different methods of mixing of the stocksolutions: diffuison-controlled mixing in space(EURECA 1992-1993 flight), convection-controlledmixing on earth and forced mixing on earth. Theduration of precipitation (crystal nucleation and crystalgrowth) was varied in a wide range.

Stock solutions 53.56 mM CaCl2 + 58.40 mM KCland 6.71 mM KH2PO4 + 46.85 mM K2HPO4 were mixedin 0.16 M KCl buffer solution under diffusion-controlledconditions in space, convection-controlled conditions onearth. In these experiments the Solution Growth Facilitydesigned and built by Oerlikon Contraves AG, Zürich,Switzerland was used (Lundager Madsen et al., 1995).

The reactor consisted of two reservoirs with calciumand phosphate solutions and the mixing chamber withthe KCl buffer solution. The holes in each wall betweena reservoir and the mixing chamber were separated by asliding valve and that was opened in orbit.

The valves were closed after 5 months of spaceflight. The complete mixing took several days and theduration of precipitation was 5 months before calciumphosphates were taken out from the crystallizationchamber.

On earth convection-controlled mixing occurred in1-2 hours and calcium phosphate samples were taken outafter 1, 2, 3 weeks, 1 month and 5 months. All solutionshad the same densities to minimize convection,temperature 37-40°C; pH was 7.4 after complete mixingbut before precipitation. A model (copy) of the SolutionGrowth Facility was used on earth at the same time ofthe flight and later to produce samples under terrestrialconvection-controlled conditions.

Forced mixing was performed during quite uncertainmixing times of about 1 – 2 seconds (fast mixing), 1 – 2minutes (moderate mixing) and several hours (slowmixing). The duration of precipitation varied from 1-2seconds to several hours and several days. For fastmixing 20 mM Ca(OH)2 and 11.97 mM H3PO4 solutions(stoechiometric composition for HAP) were used, thetemperature was 25 or 37°C, and the pH was between 6.0and 7.4. In order to study the initial state of precipitationthe crystal growth was suppressed by spraying thesolution with precipitation into liquid nitrogen.

Moderate or manual forced mixing was performedwith the same stock solutions and under the sameconditions as the previous. In addition we used very lowconcentrations of the CaCl2 and KH2PO4 + K2HPO4stock solutions when final concentrations in the mixturewere [Ca] = [P] = 1.785 mM.

Slow forced mixing of calcium chloride andpotassium phosphate solutions was carried out in astirred crystallizer with stock solutions and theconcentrations and temperature similar to those used inthe case of diffusion- or convection-controlled mixing.

Dry powders of all samples were examined by X-raydiffraction in a D/max-IIIC X-ray diffractometer (RigakuInt. Corp., Tokyo, Japan). Some specimens wereexamined by scanning electron microscopy in a JEOLJXA-8600 EPMA/SEM (Tokyo, Japan) at acceleratingvoltages of 10 and 15 kV.

A Philips CM300UT FEG (300 kV Schottky fieldemission gun, 0.65 mm spherical aberration, and 0.17 nmScherzer resolution) was used to study the crystalmorphology and the structure on the atomic level. High-resolution imaging HRTEM was performed undermicroprobe mode illumination with a 2 mm C1 aperture,50 µm C2 aperture and 100 µm objective aperture.HRTEM images were usually taken at a directmagnification of 510000 or 640000×. Selected-areaelectron diffraction (SAED) patterns were obtained inmicroprobe mode under the same illumination conditionwith a 30 µm selected-area aperture. For microdiffractionpatterns the C2 aperture was reduced to 30 µm to obtain3.5-10 nm electron microprobes with a 1.0 – 1.5 mradhalf-angle convergence.

Images were recorded with a Gatan 794 Slow-ScanCCD (Multiscan) camera with a 1024x1024 pixels/14 bitarray detector. Its associated software DigitalMicrograph3.3.1 for PC (Gatan, Inc., Pleasanton, CA) providedimage processing, including quantitative analysis andFourier filtering. Image calculation was performed withthe EMS software package (Stadelmann, 1987) by themultislice method for different values of thickness anddefocus.


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Results

Table 1 summarizes the results of this study of calciumphosphate precipitation, which include chemicalcomposition of total precipitation, crystal sizes, crystalmorphology and features of crystal structures as afunction solution mixing conditions and duration ofprecipitation. Only two crystalline phases HAP and OCPwere observed under solution conditions what were closeto physiological conditions.

An important feature of these two compounds, HAPand OCP, is the closeness of their crystallographicstructures. The set of interplanar spacings under 0.9 nmin triclinic OCP takes up all interplanar spacings ofhexagonal HAP except the 0.816 nm spacing of {011 0 }atomic planes in HAP (Figure 1). Thus, if OCP + HAPmixture formed during precipitation only this value ischaracteristic for HAP. The same that occurs for XRDidentification of the OCP phase where the most intensivepeak corresponds to the 1.878 nm (100) OCP planes.Also two other OCP reflections 11 0 (d 1 1 0=0.949 nm)and 010 (d010=0.911 nm) what are presented on thecalculated XRD pattern (Figure 1) could be characteristicfor this phase. However, in experimental patterns as to beshown below they have lower intensity and can not beclearly separated from background.

On transmission electron diffraction (TED) patternswe could not see the 100 OCP reflection due to structuraland morphological features of these crystals while theOCP reflections 11 0 and 010 are absolutely distinct. Itshould be noted also that XRD pattern in Figure 1 wascalculated for isotropic particles while in practice smalland highly anisotropic particles of only a few latticeparameters in thickness are forming. Therefore, peakintensities can differ from the calculated ones,broadening of the peaks due to small sizes and theiroverlapping takes place. As we can see, the characteristicinterplanar spacings are the largest ones for each phase.Thus, if the X-ray diffraction camera does not allow torecord low angle reflections up to 1.9 nm (about 2θ≈4°),it will not be possible to see the d100 =1.878 nm of OCPphase. This will make very difficult or even impossibleespecially in the case of small particles and mixture ofphases to assess the presence or absence of the OCPphase in precipitation using only the short interplanarspacings set in XRD phase analysis.

Space-grown samples were the first observed bySEM after taking them out from the solution and drying.SEM showed two different kinds of crystal morphologyand sizes (Figure 2a and 2b) in the specimen. The

corresponding X-ray diffraction pattern is shown onFigure 2c. The small-angle peak corresponds to the large1.88-nm interplanar spacing between the (100) OCPplanes. Also the peak for the angle 2θ=10.8° indicatesthe 0.82-nm interplanar spacing between the (011 0 )HAP planes. The small double peak immediately below10° belongs to ( 1 10 ) and (010) OCP reflections.

TEM and HRTEM were used in order to performphase analysis of individual crystals. Two calciumphosphate modifications, OCP and HAP, wereprecipitated due to inhomogeneous distribution of thesupersaturation values in the volume of the chambersince convection, the powerful mixing factor, issuppressed (Lundager Madsen et al., 1995) HAP crystalsseveral micrometers in length formed agglomerates. Thestructure of HAP crystals in details will be reportedbelow that when convection-controlled mechanism isconsidered.

One of the OCP crystals is shown in Figure 3a withthe corresponding SAED pattern (Figure 3b) taken alongthe [110] zone axis. An HRTEM image of this OCPcrystal along the [110] zone axis with the correspondingFourier transform and image calculation is shown inFigure 3c,d, e respectively. This image calculation wasperformed for OCP [110] orientation with a 0-nmunderfocus, a thickness of 1.8 nm and 0.4° tilting centerof Laue circle toward (222). Strains and bending in thecrystal lead to different mutual orientations betweencrystal parts.

The OCP crystals possess a maximum growth rate inthe [001] direction and a minimum rate in the [100]direction, i.e. along the biggest lattice parameter a.Diffusion–controlled mixing in space provides a lowersupersaturation in the crystallization systemcomparatively to earth, promoting the growth of crystalsin the competition between nucleation and growth.

The similar processes may most probably arise in thehuman body (under definite internal conditions) duringspace flying when quite large HAP crystals start to growinstead of the small and natural ones. In addition, theother modification of calcium phosphates as OCPcrystals with huge sizes appears. All these elements maydisturb the Ca dynamical equilibrium in the body whatmight lead to possible demineralization of the bonetissue.


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Table 1. Chemical composition of precipitation, morphology and crystal structure obtained under differentsolution conditions.

Solution mixingconditions

Duration ofprecipitation

Precipitationphasecomposition

Crystal sizes(maximum)

Morphology Features of structure

HAPSeveral µm in length,several tenths µm inwidth, several nm inthickness

Platelets elongated inthe c directionforming agglomerates

As a rule singlecrystalline perfectstructure

+ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅

Diffusion-controlledmixing (in space)during severaldays in theSolution GrowthFacility

5 months

OCPSeveral mm in length,tens µm in width, tensnm in thickness

Platelets elongated inthe c direction withdistinct growth stepsalong the c directionon surface

Perfect singlecrystalline structure,sometimes with smallinclusions of HAPphase.

Convection-controlledmixing (on earth)during 1-2 hoursin the SolutionGrowth Facility

1,2,3 weeks,1 month, 5months

HAP

Several tenths of µmin length, tens nm inwidth, from one tofive lattice parameters(0.82 – 4.1 nm) inthickness

Platelets elongated inthe c direction,forming agglomerates,sometimes thinplatelets orientedalong [0001]

As a rule singlecrystalline perfectstructure, sometimes agrain structure ispossible

HAP

Nanoparticles of 10-50 nm in diameter, 1-2 lattice parameters inthickness

Round nanoparticlesforming agglomerates

Single crystallineparticles and particleswith a grain structure

+ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅Fast,during 1–2seconds

1 - 2seconds OCP

Different sizes fromtenths of µm till tensµm, differentthickness

Crystals withpolygonal andirregular shape

Grain structurepossibly with largeinclusions of HAP

Moderate,during 1-2min

Severalminutes

HAP


Platelets elongated inthe c direction,forming agglomerates,sometimes thinplatelets orientedalong [0001] are alsoforming


HAP


Platelets elongated inthe c direction,forming agglomerates


+ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅

Forc

ed m

ixin

g (o

n ea

rth)

Slow,during 30hours

Several days

OCP

Several hundreds µmin length, several µmin width, tens nm inthickness

Platelets elongated inthe c direction withoutdistinct growth stepsalong the c directionon the surface

OCP matrix containsvery large HAP areas,interfaces and grainboundaries


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Figure 1. Comparison of calculated X-ray diffraction spectra of pure HAP and pure OCP phases for Cu Kαradiation with a gaussian line profile of ∆(2θ)=0.1° width.

Agglomerates of HAP crystals are shown in Figure4a and in the enlarged image (Figure 4b) with thecharacteristic polycrystalline ring SAED pattern (Figure4c). Space-grown and terrestrial HAP crystals differfrom each other in sizes: the latter are, at least, 1-1.5orders of magnitude less in length. As a rule, we observeelongated HAP platelets however, in terrestrialspecimens quite symmetrical hexagonal HAP plateletsare also presented although not very often (Figure 4 b).Figure 5a shows a HAP HRTEM image with thecorresponding Fourier transform (Figure 5b) and imagesimulation (Figure 5c) taken along the [ 2 110] direction.Image calculation was performed with a defocus valueof 44 nm for a crystal thickness of 0.9 nm. HAP crystalsare elongated in the c direction along the smallest latticeparameter (as well as OCP crystals) with its (001) planesperpendicular to a long crystal edge. A grain structure isoften observed for the terrestrial HAP crystals.

Calcium phosphate precipitation consisting only ofthe HAP phase with submicron crystal sizes can be

obtained at convection-controlled mixing of solutionswith proper duration of mixing and precipitation and atforced mixing of the stock solutions if precipitationoccurred for more than 5 minutes. The corresponding X-ray diffraction pattern for the HAP phase with thecharacteristic interplanar spacing 0.82 nm between{ 011 0 } HAP planes is shown in Figure 6.

The most striking results are obtained for specimensgrown at fast mixing of the stock solutions during 1-2seconds of precipitation and growth. The X-raydiffraction pattern from one of these samples is shown inFigure 7. Generally speaking, it is impossible to obtainreliable information on chemical composition ofprecipitation from such kind of spectrum. Only a smallpeak in the area of low angle scattering may suggest thepresence of the OCP phase. The weak peaks at theposition of the characteristic reflection of HAP couldindicate the possible existence of this phase too.


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Figure 2. SEM images of different calcium phosphate crystals obtained at diffusion-controlled mixing of thestock solutions in space (a, b), X-ray diffraction spectrum from space grown specimens, which indicatespresence of two phases OCP and HAP (c).

Figure 3a and 3b. TEM image of the space-grown OCP crystal (a) with the SAED patterntaken along the [110] direction (b).

The presence of these two phases in theprecipitation can only be ascertained by TEMobservations. Polygonal OCP crystals are shown inFigure 8a together with its SAED pattern taken along the[ 113 ] zone axis (Figure 8b). Because of the highinstability it was impossible to obtain good qualityHRTEM images from polygonal OCP crystals. RoundHAP nanocrystals are forming agglomerates (Figure 8c).The whole large agglomerate gives a SAED pattern withtwo (strong and weak) diffuse rings (Figure 8d) whilethe microdiffraction pattern obtained from an area ofabout 5 nm on the edge of this agglomerate (Figure 8e)proves the crystalline character of the particle. HRTEMimage (Figure 9a) with the corresponding Fouriertransform (Figure 9b) demonstrates high crystallinity ofthe nanoparticle, which are about 50 nm in diameter. Acalculated image of this HAP nanocrystal assuming a2.5-nm thick crystal at 33-nm focus is inserted in thecorresponding filtered image (Figure 9c). Such kind ofthe nanocrystals behaves a finer grained structure. X-raydiffraction analysis failed to reveal two phases inprecipitation and to indicate the true state and nature ofmatter because of the small sizes of HAP crystals and


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small amounts of the OCP phase. Even electrondiffraction (SAED) pattern taken from a large area of asample can be misleading. Thus microdiffraction withHRTEM possess an incontestable advantage in

investigation of small particles. This aspect is reportedin detail in a recent article published elsewhere(Suvorova and Buffat, 1999).

Figure 3c,d and e. HRTEM image of the space-grown OCP crystal along the [110] direction (c), Fouriertransform (d) and image simulation performed with a defocus value of 0 nm for an assumed thickness of 1.8 nmand 0.4° tilt (e).


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Figure 4. TEM image of terrestrial HAPcrystals obtained under convection-controlledgrowth conditions (a), enlarged image of theHAP crystals where [0001] oriented HAPcrystals are indicated by arrow (b),polycrystalline ring SAED pattern with HAPinterplanar spacings (c).


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Figure 5. HRTEM image of the HAP crystal (a) with the corresponding Fourier transform (b) and imagesimulation (c) performed with a defocus value of 44 nm for [ 2 110] zone axis and a crystal thickness of 0.9 nm.


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Figure 6. X-ray diffraction pattern for HAP crystals obtained at convection-controlled growth conditions orduring several minute precipitation after forced (fast or moderate) mixing of the stock solutions.

Figure 7. X-ray diffraction pattern from calcium precipitation obtained at fast (1-2 s) mixing of the stocksolutions and 1-2 seconds of precipitation.

Figure 8. TEM images of precipitation obtained after fast (1-2 s) mixing of solutions and 1-2 seconds ofprecipitation: polygonal OCP crystal (a) with the corresponding SAED pattern taken along the [ 113 ] zone axis(b), TEM image of round HAP nanocrystals (c) with SAED diffuse pattern obtained from the wholeagglomerate (d) and crystalline microdiffraction pattern obtained on the edge of this agglomerate with 5-nmelectron probe (e).


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Figure 9. HRTEM image of the HAP nanocrystal (a) with the corresponding Fourier transform (b) and imagecalculation shown on filtered image performed with a defocus value of 33 nm for [ 5 410 ] zone axis and acrystal thickness of 2.5 nm (c). By courtesy of J Microsc (Suvorova and Buffat, 1999)

When the duration of the precipitation exceeds fiveminutes, the OCP crystals observed in fast precipitation(1-2 s) dissolved and disappeared, while HAP crystalscontinued their growth till they reached submicron sizes.We should notice that this growth experiment goes outof the frame of biomineralization conditions. However,it shows the initial stage (or almost initial) of formation

(nucleation and growth) of calcium phosphate crystals inan environment close to physiological. Theseobservations demonstrated that there is no need to usecrystallographic undefined terms like “amorphouscalcium phosphate” or “tricalcium phosphate” todescribe the crystal growth mechanism. Clarifying of thegrowth mechanism and all possible constituents of


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precipitation is very important for direct and rationalsynthesis of HAP based bioimplants.

Very slow mixing of the stock solutions (30 hours)in a stirred crystallizer and growth during several daysresulted in a mixture of submicron-size HAP crystalsforming agglomerates and of OCP crystals elongated inthe c direction of hundreds micrometers in length. TheseOCP crystals are thinner and shorter than the space-grown OCP ones. Another very important differencebetween these crystals lies in their structure: terrestrialOCP crystals include large areas of the HAP phase while

the spatial ones almost don't. Figure 10a shows a TEMimage of a terrestrial elongated OCP crystal with thecorresponding SAED pattern (Figure 10b) taken along[110] zone axis. This crystal is quite homogeneous inthe thickness. The HRTEM image and thecorresponding FFT (Figure 10c) indicate the presence oftwo phases in the form of an OCP matrix containingHAP inclusions with the following orientationrelationship (0001) HAP // (001) OCP and [ 2 110] HAP //[110] OCP.

Figure 10. TEM image of the elongated terrestrial OCP crystal grown at slow forced mixing of the stocksolutions (a) with the corresponding SAED pattern taken along the [110] direction (b), HRTEM image of theterrestrial OCP crystal (c) and the FFT indicating presence of the OCP and HAP phases in one crystal.

Since the space-grown OCP crystals almost do notinclude any second phase and, obviously, no internalboundaries or interfaces, therefore they are quite stableunder the electron beam than the terrestrial ones, whichare destroying very quickly. A full comparison betweenspace-grown and terrestrial OCP crystals and theorientation relationship between HAP and OCP phasesin the OCP matrix are performed recently (Suvorova etal., 1998; Suvorova and Lundager Madsen, 1999). HAPcrystals have the same morphology, sizes and real andatomic structure like crystals obtained at convection-controlled mixing of the stock solutions.

This terrestrial growth experiment can represent asimplified model of bone tissue demineralization onearth, for instance during long body immobilization. TheHAP crystals formed under normal gravity conditionsare typically less than one micrometer in length and donot behave the unusual large size of space- growncrystals. The presence of OCP crystals as a second phasemay disturb the calcium dynamical equilibrium in thebody. It appeared probably due to local fluctuations ofthe heterogeneous supersaturation in the system and itsinability to dissolved quickly. The OCP crystal sizes andlowered pH value might be a reason of the storage ofthese crystals in the body.


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Local supersaturation values play the key role information of calcium phosphate crystals. In turn, it isstrongly influenced by the growth condition duringcrystallization. We are now in process of studying thecorrelation between supersaturation and growth solutionconditions on the one hand and precipitation features onthe other hand.

Conclusions

SEM, X-ray diffraction, transmission electrondiffraction and HRTEM were used for investigation ofcalcium phosphate precipitation obtained from aqueoussolutions with physiological characteristics. The effectof different growth conditions including diffusion-controlled mixing of the stock solutions, convection-controlled mixing and forced (fast, moderate and slow)mixing of the stock solutions were studied. The durationof precipitation plays a very important role for chemicalcomposition of calcium phosphate precipitation andmorphology, sizes and structure of individual crystals inthe precipitation. Under given conditions we observedthe formation of OCP and HAP crystals. Perfect andlarge (up to several mm in length) OCP crystals cangrow at diffusion-controlled mixing of the stocksolutions (in space) as well as large (up to severalmicrometers in length) HAP crystals. Convection-controlled mixing during 1-2 hours for several weeks ofprecipitation led to formation of HAP crystals ofsubmicron sizes. Forced mixing of solutions during 1-2seconds and 1-2 seconds of precipitation controlled byspraying into liquid nitrogen resulted in the formation ofa mixture of round HAP nanocrystals and polygonal orirregular OCP crystals. When ripening was allowed tocontinue for several minutes, the OCP crystals dissolvedwhile HAP crystals continued to grow till they reachsubmicron sizes. Slow forced mixing of the stocksolutions with precipitation during several days providedagain a mixture of submicron HAP crystals andelongated OCP crystals. In this case the terrestrial OCPcrystals were thinner and shorter than the space-grownOCP ones. In addition, they included in their structurenumerous and big areas of the HAP phase.

Eventually, compared to the other techniques likeSEM and X-ray diffraction, electron microdiffractionand HRTEM bring unequaled information on the state,crystallinity, chemical composition, structure anddefects even in the smallest particles and localinformation in larger ones or in agglomerates.

Acknowledgments.

Authors highly appreciate the collaboration withProf. H. E. Lundager Madsen (Denmark, Chem.

Department, Royal Veterinary and AgriculturalUniversity) and Prof. I. V. Melikhov and Dr. V. F.Komarov (Russia, Moscow State University, Chem.Department) who provided to us the calcium phosphatespecimens. Financial support and attention fromContraves Space AG (Zürich, Switzerland), andpersonally of Mr. H.Schneiter is gratefullyacknowledged. We are also thankful to Dr. M. Cantoniand Mr. G.Peter from EPFL for scientific and technicalassistance.

References

Brown W (1962) Octacalcium phosphate andhydroxyapatite. Nature 196: 1048-1050.

Fernandez E, Gil FJ, Ginerba MP, Driessens FCM,Planell JA, Best SM (1999a) Calcium phosphate bonecements for clinical applications. Part I: Solutionchemistry. J Mater Sci: Mater Med 10: 169-176.

Fernandez E, Gil FJ, Ginerba MP, Driessens FCM,Planell JA, Best SM (1999b) Calcium phosphate bonecements for clinical applications. Part II: Precipitateformation during setting reactions. J. Mater Sci: MaterMed 10: 177-183.

Kay MI, Young RA (1964) Crystal structure ofhydroxyapatite. Nature 204: 1050-1052.

Lundager Madsen HE, Christensson F, Polyak LE,Suvorova EI, Kliya MO, Chernov AA (1995) Calciumphosphate crystallization under terrestrial andmicrogravity conditions. J Crystal Growth 152: 191-202.

Stadelmann P (1987) EMS – A software package forelectron diffraction analysis and HREM imagesimulation in materials science. Ultramicroscopy 21:131-146.

Suvorova EI, Buffat PA (1999) Electron diffractionfrom micro- and nanoparticle of hydroxyapatite. JMicrosc 196 Pt 1: 46-58.

Suvorova EI, Lundager Madsen HE (1999)Observation by HRTEM the hydroxyapatite-octacalciumphosphate interface in crystals grown from aqueoussolutions J Crystal Growth 198/199: 677-681.

Suvorova EI, Christensson F, Lundager Madsen HE,Chernov AA (1998) Terrestrial and space-grown HAPand OCP crystals: effect of growth conditions onperfection and morphology. J Crystal Growth 186: 262-274.

Discussion with Reviewers

P. Layrolle: What are the experimental conditions forconvection-controlled mixing and forced mixing. Whatkind of reactor has been used for mixing calcium andphosphate solutions?


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Authors: Convection-controlled experiment wasperformed with the same stock solutions at the sametime like the space experiment and without stirring.Engineering model (copy of the space facility) of theSolution Growth Facility used on earth. The referencesare {Lundager Madsen HE et al.(1995); Suvorova EI etal. (1998)}. Forced mixing is the stirring of thesolutions.

P. Layrolle: The authors observed the preferentialgrowth of OCP crystals along the direction [001] oralong an axis by the effect of microgravity. It is wellknown that microgravity favors crystal growth ratherthan second nucleation as compared to normal gravity.However, the unusual growth of OCP crystals along[001] direction is not sufficiently discussed. How theauthors can explain this preferential orientation.

Authors: Microgravity conditions favor crystal growthrather than second nucleation as compared to normalgravity and we already showed this in the above-mentioned references. Growth of the OCP crystals alongthe [001] direction is not unusual under both spatial andterrestrial conditions as it was already mentioned inprevious works on earth grown samples (see forexample Brown et al., 1962). Irregular polygonal shapewere observed in OCP crystalline platelets prepared i) insolutions with very low concentrations (1.785 mMCaCl2) when the stock solutions were mixed with veryslow speed and ii) at very fast mixing of the stocksolutions.

P. Layrolle: The authors said that a similar processmight arise with biological apatite in the body duringspace flying. The conditions of their in vitro experiments(saturated calcium phosphate solution) as compared tobody fluids containing lower ionic concentrations andproteins are not exactly the same. It is not advisable totranspose those in vitro observations intobiomineralization processes under microgravity unlessthe discussion is supported by appropriate references onbone formation in space. The proteins present in bodyfluids might inhibit growth of apatite crystals and theauthors have not discussed this point.

Authors: The space and control earth experiment oncalcium phosphates growth showed two significantresults: the difference in size of crystals and differencein phase composition of the precipitation. An importantquestion is to understand why astronaut's bones sufferfrom demineralization during prolonged stays in space.Several factors may affect the processes involved in thebiological activities in space. The purpose of the workon space grown Ca-phosphate is to decide whether areduced gravity may induce a change in this type ofchemical reaction and should be considered in furtherstudies. A simplified model system of solutions leading

to calcium phosphate crystals formation under humanbody temperature and pH was deliberately chosen toinsure experimental reproducibility and clearinterpretation in matter of crystal growth kinetics, evenif definitely these conditions are not exactly the samelike in human body as well as the human bodies differfrom each other. Based on our results we propose that bysimilarity the processes in human body may be modifiedby micro gravity. Small changes in mineral density ofsome kinds of bone tissue were shown to result indramatic shift of mechanical properties. The loss of bonemass is an important factor but not the alone cause ofbone destruction. Possible but unknown changes incomposition and microstructure of bones wereconsidered as important factors {Oganov VS et al.,(1997); Morey-Holton ER et al., (1991)}. Alsoredistribution of calcium phosphates was observed whensome bone tissues were loosing their mineral componentwhile vertebras were characterized by a hyper-mineralization with the simultaneous decreasing theirmechanical strength {Dickenson RP et al., (1981)}

P. Layrolle: As the opposite of Posner’s observations,the authors stated that OCP or HAP crystals are directlyprecipitating in water without the transient formation ofamorphous calcium phosphate. This statement should becarefully discussed in regards to the Posner’s theory ofamorphous calcium phosphate (ACP) formation prior toHAP.

Authors: In 1965 A.S. Posner and collaborators {EanesED et al., (1965)} obtained X-ray diffraction patternsfrom (apatitic) calcium phosphates, some with peaks("crystalline"), some without any peaks told to comefrom “non-crystalline” (apatitic) calcium phosphates.This last pattern became the base of their theory on“amorphous calcium phosphate, ACP”. We also haveobtained such kind of patterns both from X-raydiffraction experiment (Figure 7) and from selected areaelectron diffraction (Figure 8d) where there are nodistinct reflections. The ability of these techniques toindicate the true state and nature of matter has to bequestioned because of the very small size of HAPcrystals (a few monolayers in thickness) and overlappingreflections. Indeed our HRTEM images andnanodiffraction patterns showed everywhere thecrystalline character of this kind of samples. Thus ourobservation did not confirm the Posner's ACP previoustheory.

P. Layrolle: Inclusion of HAP phase into OCP crystalsis observed. As half of the OCP lattice is related to thehexagonal P63/m HAP lattice, epitaxial crystal growthcan be envisaged. Nancollas and coworkers have studiedthe epitaxial growth of HAP onto OCP by using the


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constant composition method. The authors shoulddiscuss their observations in regard to the literature.

Authors: Based on the very close similarity of HAP andOCP lattices Nancollas and coworkers concluded to theepitaxial overgrowth of HAP on OCP crystals{Koutsoukos PG et al., (1981); Nelson DGA et al.(1986)}. However, the crystallographic orientationrelationships between two phases were not given. Thiswas first done in {Suvorova EI et al. (1999)}. It shouldbe noted that epitaxial overgrowth does not come intoconflict with the possibility to form the HAP inclusionsin the OCP matrix. The questions how OCP and HAPnucleates and growth–in relation with formation ofinclusions – still requires some more investigations intwo directions of growth mechanism and structure of theboundary. It will be reported in a forthcoming paper.

J.M. Bouler, G. Dacolsi: The bibliography is toolimited to the authors. The reference of analogy of OCPand HAP are too old. There are others references morerecent in particular using HRTEM (Cuisinier, Voegel,Brès…).

Authors: The references to {Kay MI et al., (1964);Brown W, (1962)} were chosen because they refer to theearly works and give the atom coordinates for HAP andOCP phases that are needed for the electron microscopyimage calculation in our work. These references werealso used for the same purpose in all later works,including Cuisinier, Voegel, Brès. In addition, it shouldbe added that these last authors described mostly theresults of studies devoted to human enamel {Brès EF etal., (1990); Brès EF et al., (1985)} or chicken bones{Cuisinier FJG et al., (1995)}.

J.M. Bouler, G. Dacolsi: The term “biomineralization”does not appear to me as fully appropriate in the title:many important factors involved in biomineralizationsuch as proteins and soluble factors are ignored in thisexperience.

Authors: We use the term of biomineralization in thesense given by G.H. Nancollas, W.E. Brown, F.C.M.Driessens, M.J. Glimcher, A.S. Posner in the specialissue of Journal of Crystal Growth (1981) 53, N1entitled “Biological mineralization”, Ed. G.H.Nancollas. This volume included the study of calciumphosphate formation and characterization underconditions close to physiological (T=37°C, pH=5.5-7.5,dilute solutions) with and mostly without organiccompounds. Some authors called these conditionpseudo-biomineralization or pseudo-physiological{Onuma K and Ito A, (2000)}.

J.M. Bouler, G. Dacolsi: As nucleation and crystalgrowth in the human body occur under the control of theproteins, it is not so sure that observed phenomena on

crystal size in vitro arise in vivo. Furthermore even inspace, body fluids are perpetually refreshed and thisfluid circulation leads probably to convection.

Authors: Withdrawal of gravity-dependant hydrostaticforces led to redistribution of liquid (plasma and blood)in the body from the bottom to the top andapproximately 2 liters of liquid moves out from the legsto upper body {Charles JB, et al., (1997); Egorov A, etal., (1988)}. This is a pure physical event. Among thenumerous reactions of living organisms, includingprotein-controlled the following changes due tomicrogravity can be observed in: i) mineral balance{Lutwak L et al., (1969)}, ii) electrolyte-fluidmetabolism {Grigoriev AI (1983)}, iii) volume of legs{Buckey JC (1988)}, iv) pulse and cardiovascularparameters {Charles JB et al., 1997}, v) venous pressure{Kirsch et al., (1984)}. All these modification can leadto deep perturbation of the natural calcium phosphatesupersaturation and its local distribution. The question iswould the protein reaction be so fast in order to continuethe control of all these processes and what is the firststep in this sequence.

J.M. Bouler, G. Dacolsi: The possible demineralizationof the bone tissue in space is also due to this absence ofthe gravitational mechanical strength which displace thecellular bone remodeling equilibrium.

Authors: We agree that the absence of load on bone hasmost probably also an effect on bone structure. Howeverin this complex situation it is mandatory to proceed stepby step. We were mostly interested here in possiblephysico-chemical processes in atomic scale occurring incalcium phosphate systems. First of all it concerned thepossible effect of micro-gravity and the methodology toassess the exact determination of phase composition,state of matter, structure, morphology and sizes ofparticles what in turn can construct bone cells.

Additional References.

Brès EF, Barry J.C., Hutchison JL (1985) High-resolution electron microscope and computed images ofhuman tooth enamel crystals. J. Ultrastruct Res 90: 261-274.

Brès EF, Voegel J-C, Frank RM (1990) High-resolution electron microscopy of human enamelcrystals. J. Microsc 160: P.2, pp.183-201.

Brown WE, Smith JP, Lehr JR, Frazier AW (1962)Crystallographic and chemical relations betweenoctacalcium phosphate and hydroxyapatite. Nature 196:1050-1055.

Buckey JC (1988) Deep venous contribution tohydrostatic blood-volume change in the human leg.Amer J Cardiol. 62: N7, 449-453.


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Charles JB, Frey MA, Fritsch-Yelle JM, Fortner JW(1997) Cardiovascular and cardio- respiratory functions,In: Man in space flight. Huntoon CL, Antipov VV,Grigoriev AI (Eds). American Institute of Aeronauticsand Astronautics, Washington. D C and RussianAcademy of Sciences, Moscow “Nauka”. Pp.109-149.

Cuisinier FJG, Steuer P, Brisson A, Voegel JC(1995) High resolution electron microscopy of crystalgrowth mechanisms in chicken bone composites. JCrystal Growth 156: 443-453.

Dickenson RP, Hutton WC, Stott JRR (1981) Themechanical properties of bone in osteoporosis. J. Boneand Joint Surg 63: N 2, pp.233-238.

Eanes ED, Gillessen IH, Posner AS (1965)Intermediate states in the precipitation ofhydroxyapatite. Nature 208: 365-367.

Egorov A, Anashkin O, Itsechovskiy O (1988)Medical investigation of results obtained in 125-dayflight on Salyut-7 and Mir orbital stations. Physiologist.3: N4, S1-S3.

Grigoriev AI (1983) Correction of changes in fluid-electrolyte metabolism in manned space flights. Aviat.Space and Environ Med 54: N4, 318-323.

Kirsch KA, Haenel F, Rocker L (1984) Venouspressure in man during weightlessness. Science 225:218-219.

Koutsoukos PG, Nancollas GH (1981) Crystalgrowth of calcium phosphates. J Crystal Growth 53: 10-19.

Lutwak L, Whedon GD, La Chance PH (1969)Mineral, electrolyte and nitrogen balance studies of theGemini 7 fourteen days orbital space flight. J Clin.Endocrinnol Metabol 29: N9, 1140-1156.

Morey-Holton ER, Arnaud SB (1991) Skeletalresponses to space flight. In: Advances in space biologyand medicine, vol. 1. Bonting SL (ed). N.Y.: JAI press.1:37-69.

Nelson DGA, Salimi H, Nancollas GH (1986)Octacalcium phosphate and apatite overgrowths: Acrystallographic and kinetic study. J Colloid. InterfaceSci 110: 32-39.

Oganov VS and Schneider VS (1997) Bone system.In: Man in space flight. Huntoon CL, Antipov VV,Grigoriev AI (Eds). American Institute of Aeronauticsand Astronautics, Washington. D C and RussianAcademy of Sciences, Moscow “Nauka”. Pp.449-451.

Onuma K and Ito A (2000) Investigation of crystalgrowth mechanism for hydroxyapatite. J Mineral SocJap 29: 17-30.

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