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Page 1: Conversion of the Echinoderm Mellita eduardobarrosoi Calcite Skeleton into Porous Hydroxyapatite by Treatment with Phosphated Boiling Solutions

Journal of Materials Synthesis and Processing, Vol. 7, No. 4, 1999

Conversion of the Echinoderm Mellita eduardobarrosoiCalcite Skeleton into Porous Hydroxyapatite by Treatmentwith Phosphated Boiling Solutions

M. A. Araiza,1,3 J. Gomez-Morales,2,4 R. Rodriguez Clemente,2 and V. M. Castano3

The influence of the boiling solution technique (a nonhydrothermal wet treatment) in the formationof hydroxyapatite (HA) from a novel source, an echinoderm known as Mellita eduardobarrosoi,whose skeleton contains calcite as an inorganic constituent, was investigated. The experimentalplan explores the conversion using a batch boiling system composed of KH2PO4 + KOH aqueoussolutions at initial pH's of 8, 9, 10, 11, and 12, where granules of skeleton were dispersed to yieldPC4/CaCO3 ratios of 1.0, 1.5, and 2.0. Chemical and X-ray diffraction analysis of the resultingmaterial showed that the conversion of calcite by this technique is almost always higher than 70%,pH's 10 and 11 yielding the highest conversion. Depending on the operating conditions, the obtainedmaterial is a mixture of the original calcite and HA of varying stoichiometry and composition.Nevertheless, the interconnected porosity is preserved.

KEY WORDS: Mellita eduardobarrosoi; calcite skeleton; conversion; hydroxyapatite; boiling system.

1. INTRODUCTION

The synthesis of HA uses several methods and avariety of sources as starting materials. The final prod-ucts can be generally named as calcium phosphate com-pounds (CPC) [1]. Among the CPC there is one kind,used as biomaterial, produced from natural sources suchas corals, shells, and skeletons of algae, which profitstheir particular initial features of porosity and chemi-cal composition. A specific class of coral (Porites) hasbeen used as raw material to obtain HA [2]. Its skeletonis composed of calcium carbonate (CaCO3), with ara-gonite as the crystalline phase. This skeleton is treated

1 Division de Estudios de Posgrado e Investigacion, Facultad de Odon-tologfa, UNAM, Circuito Institutos s/n, Cd. Universitaria, Coyoacan,Mexico D.F., C.P. 04510.

2Institut de Ciencia de Materials de Barcelona (CSIC), Campus UAB,08193 Bellaterra, Spain.

3 Instituto de Fisica, UNAM, Apartado Postal 20-360, Mexico D.F.,0100.

4 To whom correspondence should be addressed.

with phosphated solutions under hydrothermal condi-tions (high temperatures and high pressures) [3], to trans-form their chemical composition into HA, but maintainits porous microstructure. This hydrothermal process isthe main way actually used to promote the conversionof calcium carbonate into HA, producing a material withaccepted clinical use not only from corals (Interpore) butfrom calcitic algae as raw material to produce a HA use-ful as an implant material in a host environment [4].

The ultimate goal of an implant material is to obtaina substitute that matches the biological properties of nat-ural bone [5]. In this context, we have studied a new rawmaterial, namely, the skeleton of an echinoderm iden-tified as Mellita eduardobarrosoi ("sand dollar" in theGulf of Mexico). This skeleton is composed of calciumcarbonate, variety calcite [6], and has a porous structurewith connected porosity. This study reports the use of anonhydrothermal wet process for the transformation ofthe calcite skeleton of this echinoderm into porous HA,using phosphated solutions in a boiling system. The boil-ing solution method, widely used for the crystal growth

2111064-7562/99/0700-0211$16.00/0 © 1999 Plenum Publishing Corporation

Page 2: Conversion of the Echinoderm Mellita eduardobarrosoi Calcite Skeleton into Porous Hydroxyapatite by Treatment with Phosphated Boiling Solutions

212 Araiza, Gomez-Morales, Clemente, and Castano

of phosphates [7], has features of constant temperature(boiling temperature) and atmospheric pressure as oper-ating conditions; this is the first time, to our knowledge,that it has been used to obtain porous HA from naturalsources.

2. MATERIALS AND METHODS

2.1. Boiling System

The solid raw material was the skeleton of Mel-lita eduardobarrosoi, collected from the northern Coastof Gulf of Mexico, crushed and sieved; it is chemi-cally composed of >99.0% calcium carbonate in theform of calcite. The material was cleaned by immersionin 15% sodium hypochlorite for 15 days, to eliminateany organic material. Later, it was washed with distilledwater and dried in an oven at 100°C for 2 days. Twograms of granular material with particle sizes of between2 and 4 mm was used in the runs. A batch boiling system(Fig. 1) consisting of a glass pyrex reactor, heated witha heating blanket and provided with a refluxing system,was filled with 750 ml of K2HPO4 + KOH (Merck Inc.,Germany) solutions at different pH's (8-12) and over-all P/CaCO3 ratios of 1.0, 1.5, and 2.0. The system waskept boiling (T ~ 100°C) during 96 and 192 h undercontinuous N2 flow. At the end of the experiment thesamples were washed with deionized water and acetone,then dried in an oven at 90°C for 2 days.

2.2. Characterization of the Resultant Solid

The solid phase composition of the treated sampleswas detected by a qualitative X-ray powder diffraction(XRD) using a Rigaku Rotaflex RU-200B diffractome-ter (CuKa, X = 1.5418 A) in the range 5° < 26 < 60°.A quantitative analysis by XRD, aimed at calculatingthe percentage of calcite transformation into HA as afunction of the opperating conditions, was also done.For the latter purpose, samples of the calcite skeleton(>99%; calcite*) and HA*, obtained by complete trans-formation of the skeleton under hydrothermal condi-tions, were used as reference materials. All samples wereground with an automatic mortar grinder, to ensure opti-mum particle size and homogeneity. To avoid preferredorientation, a free-falling method of sample preparationwas employed. The spectra were recorded in the interval25° < 26 < 35° at a scan speed of 1°/min. The analysiswas done following the matrix-fluxing method of Chung

[8] for a binary mixture. In this method the percentagecomposition can be obtained from a regular XRD scanof the binary mixture, applying the formula:

were XHA is the percentage composition of HA, KHA,and Kc are the reference intensities of HA and calcite,and IC and IHA the intensities of the strongest line ofcalcite (104) and the (002) line of HA in the XRD spec-trum of the mixture (see Fig. 3). As the strongest lineof HA (211) is very near (112) and (300), for quanti-tative analysis the intensity of the most isolated (002)line seems more suitable. For example's sake, Eanes andPosner [9] used the HA (002) line to study the conver-sion of noncrystalline calcium phosphate to crystallinehydroxyapatite. Following the matrix-flushing method,when no matrix effects are present, the weight ratio-to-X-ray intensity ratio plot is always a straight line passingthrough the origin. The slope of this line K = ( K H A / K c )can be calculated without using the reference intensities.It is simply the corresponding intensity ratio of a 50/50%mixture of the same two components, which in this case

Fig. 1. Batch boiling system.

Page 3: Conversion of the Echinoderm Mellita eduardobarrosoi Calcite Skeleton into Porous Hydroxyapatite by Treatment with Phosphated Boiling Solutions

Conversion of the Mellita eduardobarrosoi Calcite Skeleton into Hydroxyapatite 213

are the reference materials calcite* and HA*. The detec-tion limits and precision of this method are about 0.5wt%, but they depend on the nature of the sample.

Chemical analyses of the samples were performedto compare the conversion percentages by both chemi-cal and XRD methods. The percentage composition ofC was determined with a precision of 0.3 wt% using aCarlo Erba EA-1108 elemental analyzer. This instrumenthas a combustion chamber working at 1200°C under anCh atmosphere. The Ca and P contents (ppm) were mea-sured by dissolving about 2 mg of sample in 25 ml of10% HNO3 and analyzing the solution by induced cou-pled plasma spectroscopy (ICP) in an emission ThermoJarrel Ash, Marck IV spectrometer. This instrument usesa radiofrequency source (3 kW at 27.12 MHz) and Arplasma. The precision in the Ca and P analysis was bet-ter than 2 and 1.5%, respectively.

Thermogravimetric analyses of the starting material(calcite*) and some of the final products were made ona Perkin Elmer TGA7 thermogravimetric analyzer, pro-vided with a standard furnace allowing operation fromambient temperature to 1000°C. As a heating elementit employs a Pt resistance embedded in a ceramic A12O3

liner. The precision of the thermobalance is 0.1 ug. Also,the authors performed complementary calcination exper-iments at 1000°C during 24 h. At the end of the cal-cination the furnace was cooled at a rate of 200°C/h.Then the ignition products were characterized by XRD.According to other authors [10], when nonstoichiomet-ric HA is sintered at 1000°C, a mixture of phases isobtained. This mixture is composed of HA + calcia(CaO) when Ca/P >1.667 or HA + B-tricalcium phos-phate [Ca3(PO4)2; B-TCP] when Ca/P <1.667.

The presence of functional groups (PO4) and (CO3)in samples was detected by a FT-IR Nicolet 710 spec-trometer (laser He-Ne; X = 632.8 nm) observing theabsorption bands at 605 and 1420 cm-1, respectively.The material was dispersed in KBr and the spectra wererecorded over the wavelength range 4000-400 cm-1.Finally, the texture was observed by scanning electronmicroscopy in a Hitachi S-570 microscope equippedwith an EDS analyzer for X-ray microanalysis (accel-eration voltage of the electron beam, 20 kV).

3. RESULTS

The ultimate goal on this work was to obtaina porous hydroxyapatite from a natural and abundantsource, preserving the connected porosity of its calcitic

Fig. 2. (a) SEM image of Mellita's skeleton prior to treatment. Notethe pores with diameters between 20-40 and 100-200 um. (b) Aftertreatment with phosphated boiling solutions.

skeleton. Figures 2a and b show, respectively, the scan-ning electron micrographs of the mellita's skeleton andof the sample obtained after treatment with phosphatedboiling solutions. It can be observed that the obtainedsample preserves the porosity.

The degree of CaCO3 transformation into HA wascalculated by a quantitative X-ray diffraction analysisand also by chemical analysis using the data of per-centage of residual carbon in the final solid samples, tocompare the results (see Table I). In the first approxi-mation we assumed that all residual carbon belong toresidual calcite and no other calcium phosphate phasescontaining -CO2 groups were obtained. Depending onthe operating conditions, different conversion degrees ofthe original calcite into HA were found. To analyze the

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214 Araiza, Gomez-Morales, Clemente, and Castano

Table I. Percentage Transformation (%T) of CaCO3 into Hydroxyapatite as a Function of the Initial P/CaCO3 Ratio, Initial pH(pH0), and Experimental Time in Phosphated Boiling Solutions

Sample

1m2m3m4m5m6m7m8m9m

10m11m12m13m14m15m16m17m18m19m20m

(P/CaC03)

1.01.01.01.01.01.51.51.51.51.52.02.02.02.02.02.02.02.02.02.0

pHo

89

10111289

10111289

10111289

101112

Time (h)

969696969696969696969696969696

192192192192192

%T(A)a

75.985.075.582.574.478.267.889.575.975.672.574.192.075.638.479.979.879.073.578.6

%T(B)b

75.383.565.083.673.270.757.694.575.272.072.078.988.075.229.366.575.474.089.470.7

Ignition productsc

HA, CaO, B-TCP*HA, CaO, B-TCPHA, CaO, B-TCPHA, CaO, B-TCPHA, CaOHA, CaO, B-TCP*HA, CaO, B-TCPHA, CaO, B-TCPHA, CaO, B-TCP*HA, CaO, B-TCP*HA, CaO, B-TCP*HA, CaO, B-TCP*HA, CaO, B-TCPHA, CaO, B-TCP*CaO, HAHA, CaO, B-TCPHA, CaO, B-TCPHA, CaO, B-TCPHA, CaO, B-TCPHA, CaO

aCalculated from elemental analysis of residual C. %T= 100 - (%C/12.01) x 100.08.bCalculated from quantitative XRD data using the intensity of peaks (104) of calcite and (002) of HA. Method of Chung [7].The ignition products characterized by qualitative XRD, when samples were calcined at 1000° during 24 h. B-TCP*: traces of B-TCP

(relative intensity of strongest XRD peak of B-TCP less than 5%).

Table II. Statistical Study of the Influence of the Process Parameters(initial P/CaCO3 and Initial pH's) on the Conversion Percentages

of Calcite into HA at Processing Times of 96 ha

P/CaC03

11.522*

pH89101112

<7XA)>

78.777.470.578.2*

76.676.684.076.866.7

aA

4.37.0

17.52.4*

2.76.46.93.4

16.4

<T(B)>

76.274.066.775.2*

71.173.880.381.061.3

OB

5.111.720.5

7.7*

3.29.8

11.56.0

18.2

a<T(A)> and <T(B)> are the mean conversion values obtained fromchemical analysis and DRX respectively. aA and aB are the corre-sponding standard deviations. *Values obtained at processing timesof 192 h.

influence of these operating conditions on the conver-sion degrees, we compared in Table II the mean con-version value (<T(A)> and <T(B)>) and their correspond-ing standard deviation (a) for each group of operatingconditions (PO4 :CO3Ca molar ratios and initial pH's).

From these data it can be deduced that initial pH's of10 and 11 yield the highest degree of transformation,independently of the initial PO4/CO3Ca ratio. There isnot a clear relationship between the PO4:CO3Ca ratioand conversion degrees. However, it can be observedthat raising the PO4 :CO3Ca ratio at processing timesof 96 h increases the standard deviations. This can beattributed to the increased influence of pH on the con-version values. Increasing the synthesis time from 96 to192 h (runs 16m to 20m) does not imply an increase inthe percentage of transformation, but also in this seriesthe highest conversion of calcite into HA was observedat initial pH's of 10 and 11 (Fig. 3). Even in these casesthe strongest peak of calcite (20 - 29.7°), correspond-ing to the reflection (104), does not disappear, indicat-ing that the carbonate exists as a separate CaCO3 phase.Also, it can be observed in the IR spectra (Fig. 4) thatthe broad CO3 absorption band at 1420 cm -1 persistsafter the treatment with a phosphated boiling solution.This adsorption band, however, also appears when CO3

groups are present in the lattice of carbonate apatites. ByIR analysis, we cannot distinguish the distribution of theCO3 groups between two solid phases.

An interesting observation is the poor agreement

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Conversion of the Mellita eduardobarrosoi Calcite Skeleton into Hydroxyapatite 215

Fig. 3. X-ray diffraction patterns of samples 16m to 20m showing thepeaks (104) of calcite and (002) and (211) of hydroxyapatite.

observed in most of the experiments between the trans-formation degrees calculated using data on the percent-age of residual carbon, T(A) (Table I), and those cal-culated by X-ray diffraction, T(B), disregarding pos-sible experimental errors. In these experiments usuallyT(A) > T(B). This fact can be attributed to the fol-lowing phenomena: (1) part of the transformed HA ispresent as amorphous calcium phosphate, or (2) part ofthe carbon determined in the final samples is present asCOs-apatite. According to Le Geros [1], CO3 groupstend to amorphize the hydroxyapatite. In only four exper-iments (samples 4m, 8m, 12m, and 19m) did we obtainT(A) < T(B).

On the other hand, the presence of unconvertedCaCO3 in the final products makes analysis of the HAdifficult; for example, quantitative analysis of Ca and Pperformed by the ICP technique does not give an indi-cation of the stoichiometry (Ca/P ratio) of the apatiticpart of the samples. Thus, the samples can be composedof residual CaCO3 plus stoichiometric HA or of CaCO3

plus nonstoichiometric HA. Other possibilities such asthe coexistence of calcite and carbonate-apatite also can-not be excluded.

To explore these possibilities of composition, wecompared the results obtained by chemical analysis ofC, Ca, and P with those obtained by quantitative X-raydiffraction. We used the following assumptions aboutthe possible composition of the transformed samples: (1)CaCO3 plus stoichiometric HA [Ca10(OH)2(P04)6], (2)CaCO3 plus Ca-deficient hydroxyapatite (Ca-dHA), of

Fig. 4. Infrared spectra of Mellita's skeleton, before and after treat-ment with phosphated solutions in a boiling system. The strongestchanges are observed at absorption bands 1420 and 605 cm- 1 , cor-responding to CO3 and PO4 functional groups.

composition Ca9(HPO4(PO4)5OH, and (3) CaCO3 plusan ideal carbonate-apatite (C03-HA), of compositionCa10(PO4)5(CO3)2OH, where both type A and type Bcarbonate incorporation would be present equally in thestructure. Considering the percentage composition (W)of each component (compound or functional group) inthe different mixtures, the following relationships can beobtained.

(1) For the mixture CaCO3 + Ca10(OH)2(PO4)6,

(2) For the mixture CaCO3 + Ca9(HPO4)(PO4)5OH,

(3) For the mixture CaCO3 + Ca10(PO4)5(CO3)2OH,

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216 Araiza, Gomez-Morales, Clemente, and Castano

For the sake of simplicity we consider

sent the overall chemical composition of each compoundin the mixture calculated by chemical analysis and quan-titative X-ray diffraction. This type of correlation hassome weaknesses: W(OH) cannot be measured directly,but it can be estimated in its maximum and minimumamount, relating it to the PO4 content of the stoichiomet-ric hydroxyapatite (W(OH)/W(PO4) = 0.06), carbonateapatite (W(OE)/W(PO4) = 0.035), or Ca-deficient hy-droxyapatite (W(OH)/W(PO4)) = 0.03.

We can observe in Fig. 5a that the plot of chemi-cal composition vs crystalline phase composition of mostof the samples falls on theoretical lines and in betweenlines. However, in some samples, the points fall outsidethe limiting theoretical value corresponding to the cal-cite + tricalcium phosphate mixture (Fig. 5b). The lattersituation clearly points to the existence of an amorphouscalcium phosphate [1], contributing to a high PO4 con-tent, not detected by XRD.

The results obtained in the calcination experiments(Table I) show that ignition products are composed ofCaO, HA, and tricalcium phosphate (B-TCP), exceptthose obtained from samples 5m, 15m, and 20m, whichare composed of CaO plus HA only. In the ignition prod-

The plot of W(PO4)/(W(PO4) + W(C03) + W(OH)) ver-sus W(apatite)/(W(calcite) + W(apatite)) for each mix-ture yields straight lines intercepting the origin (Figs. 5aand b). These lines represent the theoretical compositionof calcite + apatite mixtures with increased proportionsof apatite. As a reference line, we also include that cor-responding to the mixture CaCO3 + Ca9(PO4)6 (trical-cium phosphate), considered as a limiting case of eachCa-deficient apatite. This type of plot allows us to repre-

Fig. 5a. Plot of the overall chemical composition of some transformed (•) samples, expressed asthe ratio of the fractional weight W(PO4)/(WPO4) + W(CO3) + W(OH)) vs W(apatite)/(W(calcite)+ W(apalite)), calculated by using data from the chemical analysis of C, Ca, and P and the per-centage conversion in the column %T(B) in Table I.

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Conversion of the Mellita eduardobarrosoi Calcite Skeleton into Hydroxyapatite 217

Fig. 5b. Plot of the overall chemical composition of some transformed (C) and thermally treatedsamples at 250o C (O) expressed as the ratio of the fractional weight W(PO 4) / (W(PO 4) + W(CO3)+ W(OH)) vs W(apatite)/(W(calcite) + W(apatite)).

ucts obtained from samples 1m, 6m, (9-12)m, and 13m,we observed only traces of B-TCP. This thermal behav-ior confirms that we obtained calcium-deficient hydroxy-apatites in most of the runs. However, samples 5m, 15m,and 20m are composed by CaCO3 plus stoichiometrichydroxyapatite. The composition of these samples fallson the theoretical line (Fig. 5a). In the latter cases theexperiments were carried out at an initial pH of 12,where the predominant phosphate specie in solution isPO3-.

Thermogravimetric analysis of the starting materialshows that CaCO3 starts to decompose into CaO andCO2 at temperatures between 250 and 300°C, whereasthermograms of some of the treated samples show H2Olosses at 120°C and strong CO2 losses around 600°C.These results show that samples can be treated ther-mally, without decomposition of CaCO3, at temperaturesaround 250°C. This thermal treatment allows the amor-phous apatitic phase to recrystallize, while the calcitedoes not decompose, as confirmed by quantitative XRDanalysis. The results (see Fig. 5b) shows a clear displace-ment to the right of most of the points outside the upperlimit.

Finally, we observed a direct relationship betweenthe filling of surface pores by a powder material (poorly

Fig. 6. X-ray diffraction pattern of poorly crystalline apatite recoveredfrom the porous material using an ultrasonic bath. In this diagram alsoappear a peak at 26 = 29.7 , corresponding to residual calcite, and threepeaks between 26 = 17 and 22 . corresponding to the filter where theparticles were deposited for the XRD analysis.

crystalline apatite) and the increased P/CaCO3 ratio ofthe system. A P/CaCO3 ratio of 2.0 causes the highestobstruction of pores. The precipitation and deposition ofapatite over the inner and outer surfaces of the materialwere verified by X-ray diffraction of the powder recov-ered from the porous material using an ultrasonic bath(Fig. 6) and by X-ray microanalysis (Fig. 7). Traces ofSi and Mg were found in lower proportions than in theinitial raw material.

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218 Araiza, Gomez-Morales, Clemente, and Castano

more insoluble calcium phosphate. The coexistence ofboth crystalline phases can be a very convenient meansof controlling the resorbability of the material.

The analysis of the resulting material shows that theconversion of calcite by this technique is almost alwayshigher than 70% and that the three possible apatiticphases are formed. There is no clear relationship betweenthe amount and type of apatite formed and the processvariables: P/CO3Ca intitial pH, and length of the runs.It seems only that initial pH's of 10 and 11 yield higherconversion ratios.

It is interesting to note that in an early study by Royand Linnehan [3], B-tricalcium phosphate was obtainedon attempting to convert calcite into HA. These authorsrelate the presence of whitlockite to a high Mg content inthe initial raw material. However, the initial raw materialused in this study is composed of calcite, with a minorfraction of Mg, thus supporting the hypothesis of Royand Linnehan [3]. Most biomaterials present on the mar-ket produced from conversion of biological calcium car-bonate use aragonite as the starting material, and theyare reported to be composed of HA, such as in the caseof Interpore and Algipore. In other cases, they are usedas bare calcium carbonate (Biocoral).

This work represents an attempt to obtain a porousbiomaterial based in HA from an abundant biologicalsource such as porous calcite from an echinoderm skele-ton, without the use of high temperatures or pressures inthe system. The experimental conditions (pH and timeof processing) to transform CaCO3 into HA with vari-ous degrees of transformation have been determined. Theresulting calcite-apatite composite materials, with pre-served porosity, are potentially useful because they couldallow good cell penetration. The varying calcite/apatiteratio also implies a different solubility when they areplaced in a host environment. Currently, from the clinicalpoint of view, the use of pure stoichiometric hydroxy-apatite is a matter of discussion, because its low solu-bility can act as a retarding factor in the substitution ofimplant sites by osteoblast cells. Therefore, it would beconvenient to develop new biocompatible materials withvarying characteristics of porosity and solubility as mainattributes, which are matched by our products.

ACKNOWLEDGMENTS

This work was supported by Project CICYTMAT98-0976-C02-01 of the Spanish National ResearchPlan and Project CYTED VIII.6. J.G.M. and R.R.C.

Fig. 7. X-ray microanalysis of the inner surface of samples treated; Siand K trace elements are present due to the system disadvantages.

4. DISCUSSION AND CONCLUSIONS

A boiling system has been used to promote the con-version of calcite into HA. The pH evolution along thereactions was not recorded, but in some experiments,at the end of the runs the pH was measured again. Anincrease of about 1 unit with respect to the initial pH wasfound when the initial pH was 9, 10, or 11, where thepredominant phosphate species is HPO2-.

The chemical transformation suffered by the start-ing material is an ionic exchange heterogeneous reaction,which can be described by the following equation:

5 CaC03(calcite) + 3 HPO2- + 2 H2O

= Ca5(OH)(P04)3(HA) + 5 HC03- + OH~ (18)

However, this method produces an incompletetransformation. The final material is a varying mixtureof the original calcium carbonate and HA of varyingstoichiometry and composition, but the porous textureof the original material is mostly preserved. However,under certain conditions (initial P/Ca = 2), there are twosimultaneous processes: conversion of calcite into HAby ionic diffusion of the phosphate and hydroxyl ionsand local precipitation of HA in the pores by reactionof some leached Ca and the phosphate. The latter phe-nomenon is an undesirable one due to the partial obstruc-tion of pores resulting. The technological target of thisprocessing should be to keep the fine interconnectedporosity, while transforming calcium carbonate into the

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Conversion of the Mellita eduardobarrosoi Calcite Skeleton into Hydroxyapatite 219

belong to Excellence Research Team SGR97-00024,financed by the autonomous Government of the Gener-alitat de Cantalunya. M.A.A. and V.M.C. wish to thankthe Universidad Nacional Autonoma de Mexico andthe Institut de Ciencia de Materials de Barcelona fortheir support and the Direction General de PersonalAcademico de la UNAM for its postdoctoral fellowshipto M.A.A.

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