Solid State Sciences 8 (2006) 685–691www.elsevier.com/locate/ssscie

Nanocrystalline bioactive apatite coatings

Natalia Hijón a, M. Victoria Cabañas a, Isabel Izquierdo-Barba a, M. Angel García b,María Vallet-Regí a,b,∗

a Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spainb Instituto de Magnetismo Aplicado, RENFE-UCM, Apdo. 155, 28230 Las Rozas, Spain

Received 7 November 2005; accepted 19 January 2006

Available online 29 March 2006

Abstract

A study of carbonatehydroxyapatite coatings deposited by a sol–gel technique from different precursor-sols containing different water/ethanolcontent is performed. All the sols form uniform and homogeneous coatings with good adhesive strength values. The composition of sols determineboth the aging time necessary to deposit monophasic apatite coatings as well as the time interval to deposit said coatings. The thickness, porosityand roughness of the coatings are also modified as a function of the precursor-sol. After the immersion into an acellular simulated body fluidsolution, the coatings appeared to be constituted by two types of particles: nanoparticles smaller than 25 nm with a Ca/P molar ratio of 1.7,corresponding to the apatite sol–gel coating, and an other type of apatite particles with needle shape and Ca/P molar ratio of 1.4, correspondingto the mineralization process of the coatings. The results obtained indicate that a compromise among the aging time, coating roughness, porosityand thickness must be reached by using these precursor-sol for a technological development of the sol-gel technique.© 2006 Elsevier SAS. All rights reserved.

Keywords: Hydroxyapatite coatings; Sol–gel technique; Biomaterials

1. Introduction

In vivo studies of hydroxyapatite (HA) coatings have shownto promote osteoconductivity on Ti and Ti alloy substrates [1,2].These coatings have been deposited by numerous depositionmethods, and fundamentally, plasma-spray [3–5]. Consideringthat the majority of the methods require sophisticated appa-ratus, the sol-gel technique is a relatively low cost method.Besides, this method presents other advantages, such as lowsynthesis temperature, the ability to coat porous and complexsurfaces and a high control of composition, microstructure andthickness of obtained films. The quality of these coatings is de-termined by the chemical characteristics of the starting sols.In this sense, several authors [6–23] have deposited HA coat-ings by using different calcium and phosphorous precursors(Table 1) in order to control the chemical nature of the cal-cium phosphate, the porosity and the thickness of the coatings.In spite of the potential technological interest of this synthesis

* Corresponding author. Fax: +34 913941786; tel.: +34 913941861.E-mail address: vallet@farm.ucm.es (M. Vallet-Regí).

1293-2558/$ – see front matter © 2006 Elsevier SAS. All rights reserved.doi:10.1016/j.solidstatesciences.2006.01.008

method and the high number of publications in this area, the dif-ferences of coatings deposited by the use of different conditionsof precursor sol have not yet been studied.

In a previous study [16] we showed the necessity to con-trol the aging time of the precursor sol as a function of the soltemperature, in order to obtain a monophasic HA coating. Forthis purpose, aqueous solutions of calcium nitrate and triethylphosphite were chosen due to the rapid hydrolyzation of alkylphosphites and also because the presence of oxidant agents suchas nitrate anions would favor the decomposition of ethyl groupsduring the synthesis process. The purpose of the present workis to compare HA films deposited by dip-coating using differentprecursor-sols with varying ethanol/water ratios, and to estab-lish the optimum conditions for a technological developmentof the sol–gel technique applied to the coating of metallic sub-strates.

2. Experimental

2.1. Precursor sols

To prepare the starting solutions, triethyl phosphite,P(OCH2CH3)3 (Aldrich) and calcium nitrate, Ca(NO3)2·4H2O

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Table 1Some of calcium and phosphorous precursors used in the synthesis of HA coatings deposited by sol–gel technique

Reference Calcium precursor/solvent Phosphorous precursor/solvent

Brendel et al. [6] Calcium nitrate/acetone Phenyldichlorophosphine/acetone/waterRussel et al. [7] Calcium nitrate/2 methoxyethanol N -butyl acid phosphate/2 methoxyethanolHsieh et al. [8] Calcium nitrate/2 methoxyethanol Triethyl phosphate/2 methoxyethanolGoins et al. [9]a Calcium nitrate/2 methoxyethanol Diethyl phosphite/2 methoxyethanolYou et al. [10]b Calcium nitrate/methanol Triethyl phosphite/methanolHwang et al. [11] Calcium nitrate/methanol Phosphoric acid/methanolKojima et al. [12] Calcium nitrate/ethanol Triethyl phosphate/ethanolLiu et al. [13] Calcium nitrate/ethanol Triethyl phosphite/ethanol/waterGan et al. [14] Calcium nitrate/ethanol Triethyl phosphite/waterPiveteau et al. [15] Calcium nitrate/ethanol Phosphoric pentoxide/ethanolHijón et al. [16] Calcium nitrate/water Triethyl phosphite/waterCavalli et al. [17]c Calcium nitrate/water Diammonium hydrogen phosphate/waterGan et al. [14] Calcium nitrate/water Ammonium dihydrogen phosphate/waterWeng et al. [18]d Calcium glycoxide/ethyleneglycol Phosphoric pentoxide/ethanol or butanolChai et al. [19]e Calcium diethoxide/ethanol/ethanediol Triethyl phosphite/ethanolGross et al. [20] Calcium diethoxide/ethanol/ethanediol Triethyl phosphite/ethanol/ethanediolHaddow et al. [21]f Calcium diethoxide/ethanediol Triethyl phosphite/ethanediolBen-Nissan et al. [22] Calcium diethoxide or calcium acetate/ethyleneglycol/acetic acid DiethylhydrogenphosphonateTkalcec et al. [23]g Calcium 2-ethylhexanoate/ethylhexanoic acid 2-ethylhexylphosphate/ethylhexanoic acid

a Add small amount of citric acid for stabilization; b Add HCl and DCCA (Drying Control Chemical Additive); c The aged sol was diluted in ethanol

(2 : 1); d Add ethylenediamine to adjust the pH; e Add acetic acid to stabilization; f Add ethanol to modify the sol viscosity; g Add ethanol to reduce theconcentration.

Fig. 1. The preparation conditions of the different precursor sols used for thecoating deposition (*—the different water : ethanol ratios used).

(Aldrich), in a Ca/P molar ratio equal to 1.67, were used as pre-cursor solutes. These compounds were hydrolyzed/dissolved inpure ethanol (sol E), pure water (sol W and sol WD) or a mix-ture of both solvents (sol EW), according to the scheme showedin Fig. 1. Initially, the phosphorous sol is prepared by addingeither water or ethanol, and the calcium containing solutionis added afterwards. Further details of similar sol preparationwere described previously [16].

The precursor sol solutions were aged at 60 ◦C in an ovenduring different times before being used to make coatings. Thisaging temperature was chosen according to previous results ob-tained working with aqueous sols [16] which showed low agingtime for this temperature. The variation of pH and viscosity of

the sols with time were measured by using a pHmeter (ModelMetrohm 744), and a Haaker ReoStress RS75 rheometer, at ashear rate range from 1 to 200 s−1 at 20 ◦C, respectively.

2.2. Films preparation and characterization

Apatite calcium phosphate coatings have been deposited, bydip coating method, onto disc substrates of Ti6Al4V. Beforecoating deposition, substrates were polished with SiC of grit320 and 9-3-1 µm diamond paste, and washed for 5 min in anultrasound bath with distilled water, alcohol and acetone.

The metal substrates were dip coated under room conditionswith the above described precursor-sol solutions, at withdrawalrates varying from 800 to 2500 µm/s. After the deposition, thecoatings were dried at 100 ◦C (1 h), annealed in air at 550 ◦C(10 min) and then cooled to room temperature. Finally, the coat-ings were washed with ethanol in an ultrasound bath for twominutes.

A dissolution/bioactivity test of deposited coatings was per-formed. The samples were soaked in a simulated body fluid[24] (SBF) at 37 ◦C and buffered at pH = 7.4 for up to oneweek. The SBF was previously filtered and all operations werecarried out in a laminar flux cabin, avoiding bacterial contami-nation. After soaking, the coatings were removed from the fluidand washed with water and ethanol.

The crystal structure of the deposited coatings was char-acterized by X-ray diffraction (XRD) on a thin film PhilipsX-Pert MPD diffractometer using Cu Kα radiation. The molec-ular groups of the films were determined by Fourier TransformInfrared Spectroscopy (FTIR) in a Nicolet Nexus spectrome-ter using an ATR Golden Gate. The surface morphology ofthe coatings was examined by Scanning Electron Microscopy(SEM) on a JEOL 6400 instrument and by Scanning Force

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Microscopy (SFM) on a Autoprobe-cp (Park Scientific Instru-ments) working in contact mode. The Ca and P molar ratio wasdetermined by Energy Dispersive X-ray spectrometry (EDS).The thickness of the coatings was analyzed by interferencefringe technique on a spectrophotometer Schimadzu 3101 at-tached with an integrating sphere and reflectance accessory.The adhesive strength was measured by the pull out test, in ac-cordance with American society for Testing Materials (ASTM)specifications [25]: a pair of metallic jigs were bonded to thecoated and the uncoated titanium substrates, these ensembleswere joined by means of the Loctite 480 adhesive. After leav-ing the set for 1 day, for complete solidification of the adhesive,the adhesive strength of the film was measured by applying atensile stress using a universal mechanical tester MicrotestSCM3000 at a crosshead speed of 0.5 mm/s until fracture occurred.Five measurements were done for each data point.

The surfaces of the coatings, after immersion in SBF, wereanalyzed directly by XRD, FTIR and SEM. Electron Diffrac-tion (ED) and Transmission Electron Microscope (TEM) werealso performed, by using a JEOL 2000 FX electron microscope,on material scrapped from the surface coating with a metallicblade.

3. Results and discussion

In all the systems, the pH of the precursor-sol solutionsvaries with the aging time. The pH showed an abrupt decreaseat initial time periods. After that the pH decreased moderatelywith the aging time. The viscosity measurements show a New-tonian behavior in all the sols. The viscosity values, taken at ashear rate of 200 s−1, are higher for sols E and EW, 20 and15 mPas, respectively, than those found for aqueous sol (about5 mPas). That is, the solution viscosity decreases as the ethanolcontent into the hydrolysis process decreases. No viscosity vari-ations were observed with the aging time.

In the XRD patterns of the non-heat treated coatings(Fig. 2(a)) no diffraction maxima were observed (only thoseattributable to the substrate) indicating that the films wereamorphous. After heat treatment at 550 ◦C, these amorphouscoatings crystallized into different crystalline phases as a func-tion of sol composition and aging time (Table 2). For example,for an aging time of 24 hours, sols E and EW lead to the for-mation of CaCO3 (calcite) (Fig. 2(b)), films with an apatite-likestructure are obtained from sol WD (dilution 1 : 2) (Fig. 2(c)),whereas powder onto the substrate appears when sol W is used,which is not desirable in the films preparation.

Data collected in Table 2 show that the higher the water con-tent is, lower is the aging time necessary to obtain a monophasicHA coating. That is, the hydrolysis/polymerization reaction ofthe calcium–phosphorus precursors [26,27] is retarded in pres-ence of ethanol. In the same way, in presence of ethanol, theprecursor sol produces apatite layers in a wider aging timeinterval, before powder was formed onto the substrate. Con-sequently, from an industrial point of view, the best resultscorrespond to the use of aqueous sols, because HA coating canbe performed within a short period of time; however, the ag-ing time interval to produce an apatite layer is shorter, which

Fig. 2. XRD patterns corresponding to (a) non-heated coatings; coatings heatedat 550 ◦C and deposited by using (b) sol E aged 24 hours; (c) sol WD (1 : 2ratio) aged 24 hours.

Table 2Crystalline phases detected by XRD on annealed films deposited from sols agedat different times

Aging time (h) Sol E Sol EW Sol W Sol WD

6 – – HA HA24 Calcite Calcite Powder HA48 HA + Calcite HA + Calcite – HA72 HA + Calcite HA – HA96 HA + Calcite Powder – Powder

120 HA – – –144 HA – – –192 Powder – – –

The data showed from sol WD correspond to the dilution 1 : 2.

is not desirable from a practical point of view, because there isless time to make coatings with the same precursor-sol. There-fore, sol WD, should be the best candidate for reaching bothobjectives.

The FTIR spectra of the apatitic calcium phosphate coatings(Fig. 3) show characteristic absorption bands corresponding toan apatite structure [28]. Also the FTIR spectra revealed thatcarbonates were incorporated into the apatite lattice. The pres-ence of these carbonate groups can be explained consideringthe ethyl groups decomposition during the synthesis process.The absorption bands at 1446 and 1455 cm−1 correspond toCO3

2− groups incorporated at phosphate positions anions [29].Consequently, the deposited films can be considered as a car-bonatehydroxyapatite type B.

All the apatite coatings showed similar XRD patterns andFTIR spectra, irrespective of the composition of sol precursor.

During the deposition process, the substrate is immersed ina dipping solution and is withdrawn up vertically. The amount

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Fig. 3. FTIR spectra corresponding to an annealed coating deposited using thesol E aged 5 days.

of the solution dragged by the substrate depends on the solutionviscosity and the speed of withdrawal [30]. In this sense, thewithdrawal speed used for preparing the coatings depends onthe composition of the precursor-sol used to make the coatings.In sols containing ethanol (E, EW and WD) it was possible tomake coatings from 200 µm/s to the higher withdrawal speed,2500 µm/s, whereas in total aqueous sol, sol W, it was not pos-sible to increase the withdrawal speed more than 800 µm/s,because the sol does not properly adheres to the substrate. Thisfact indicates that the presence of ethanol into the sol helps todeposit thicker films, probably by the higher ethanol rate ofevaporation that allows a proper deposition.

According to that, different coating thicknesses were ob-tained as a function of precursor-sol and withdrawal speed:sol E leads to coatings with a thickness of 940 ± 20 nm whenthe speed used is 800 µm/s and 1450±5 nm for a withdrawal of2500 µm/s; the sol EW leads to thicknesses of 1000 ± 20 nm(2500 µm/s), whereas the thinner coatings were deposited byusing the less viscous sol W, which led to thicknesses of 200nm, for a speed of 800 µm/s. Coating thicknesses from 600 to100 nm are obtained from sol WD as a function of dilution em-ployed; as the water : ethanol ratio increases lower are the thick-nesses. On the other hand, working in a totally aqueous system(sol W), it was possible to repeat the coating procedure until 10times, before powder was formed onto the substrate, reachingthicknesses of about 2 µm [16]. The higher thickness observedin coatings deposited by using ethanol containing sols, whichpresent a higher viscosity, justify that the procedure could notbe applied different times, just one for sol E and a speed rate of2500 µm/s, and two times when the rate used was 800 µm/s.

The films appeared cracked or peeled indicating that a criticalthickness was exceed.

Fig. 4(a) represents a SEM micrograph of a monophasic HAcoating, deposited by using sol EW, showing the surface aspectof the coating. The surface of the substrate can also be ob-served because, during the deposition of the film by dip-coating,the clamps holding the substrate should not enter the solution.Then, there is always a substrate region which is uncoated. Thismicrograph shows that the surface morphology of the sol–gelcoating is very similar to that of the Ti6Al4V substrate. Thecoating appears uniform, crack free, dense and homogeneousand with a smooth surface (Fig. 4(b)). On the other hand, thebiphasic nature of some coatings (CaCO3 + HA) observed byXRD for low aging times, is confirmed by SEM micrograph(Fig. 4(c)) which shows the presence of two types of particleswith different relative amounts of Ca and P, according to theEDS analysis.

SiO2–CaO vitreous films deposited by dip-coating ontoTi6Al4V substrates showed different porosity as a function ofethanol content [31,32]. However, the SEM study performed onmonophase apatite coatings obtained in this work shows thatthe surfaces are practically similar; all of them seem dense andthere are no differences as a function of the sol composition.In this point, it is worth noting that during the film depositionby dip-coating a ridge is formed at the lower edge of the sub-strate, after the withdrawal from the coating bath [30]. Althoughall films deposited in this work were homogeneous, accordingto visual observations and SEM micrographs, the formation ofthis ridge is smaller when ethanol is introduced into the sol pre-cursor. Then, the presence of ethanol into the solution allowsto uniformly wet the surface of the substrate, achieving moreuniform coverage, allowing to coat larger and complex surfacesmore easily.

Fig. 5 shows the profiles, 3D images and roughness valuesobtained by the SFM study of HA obtained films deposited byusing the different precursors. The SFM images confirm thatdense HA coatings cover the Ti6Al4V substrate. These coat-ings show different values of roughness (rms) and porosity (seeprofiles), which could not be detected from SEM images, as afunction of precursor-sol composition.

The films obtained from sol E show a porous surface with apore size of 120 nm diameter and 10 nm depth and a roughnessvalue about 8 nm. When the ethanol volume decreases, for filmsobtained by using sol EW, the rms value decreases to 5.1 nm,showing a less porous surface, with a lower pore depth value,

Fig. 4. SEM micrographs corresponding to coatings deposited using sol EW aged during 72 hours (a, b); or during 48 hours (c).

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Fig. 5. SFM profiles, 3D images and roughness (rms) data corresponding to HAcoatings deposited from the different sols.

about 5 nm. In the case of the film obtained from sol W (with-out ethanol) the roughness value is 4.4 nm and the SFM studyshows a smoother surface. Films obtained from sol WD (1 : 2dilution) showed a high roughness, about 7.5 nm, and no poreswere detected.

These results show an increase in surface roughness andporosity when the ethanol concentration into the precursor solincreases. The effect of increasing the ethanol content in theprecursor-sol implies that the polymerization reaction kineticsis retarded, the viscosity of the sol increases and then, probablythis causes that the size of Ca–P containing aggregates increasesforming more porous coatings. When the ethanol is added af-ter the hydrolysis process, in films obtained from sol WD, thesituation is different, the films are rougher but not porous. Inthese sols, the polymerization reaction is produced before theaddition of ethanol, then no pores appear, but the subsequentevaporation of the solvent during the film deposition helps togenerate roughness within the film.

Although the analyzed coatings show different thicknesses,which could influence in the results obtained, we would like toremark that the SFM study of thinner coatings deposited fromsol E led to similar results of porosity to those obtained forthicker coatings by using the same sol.

The adhesion test of HA coatings deposited shows valuesabove to 20 MPa; under such stress breakage occurs in the adhe-sive, in the adhesive/coating interface or between the substrateand the metallic holder; in no case fracture of the coating is ob-served. Thus, the value of 20 MPa must be considered as a lowerlimit. No significant differences were observed in coatings de-posited from the different sols. These tensile strength adhesionvalues are comparable to those obtained for HA coatings pre-pared by other preparation methods, such as electrodeposition,plasma spray or pulsed laser deposition [33–35].

The apatitic coatings deposited by sol–gel were soaked intoSBF. Glancing-angle incidence XRD patterns obtained from thesurface of the sol–gel coatings after soaking in SBF were verysimilar to those obtained before immersion; all the diffractionmaxima can be assigned to an apatite structure. In the same way,FTIR spectra of coatings after immersion shows bands corre-sponding to phosphate and carbonate groups, similar to thoseobserved before soaking.

On the other hand, the SEM micrographs corresponding tocoatings soaked in SBF during 7 days, show that the surfaceof the films is different to that obtained before immersion. Thezone coated with an apatite sol–gel film, is now covered by anew layer of material which appears to be constituted by numer-ous needle-like crystallites (Fig. 6), similar to those observed inother bioactive systems [36].

All the HA coatings studied in this work showed the forma-tion of this new layer, independent of the precursor-sol used forits deposition. However, the size of the needle shaped crystal-lites depends on the precursor solution (Fig. 7). Films depositedat 2500 µm/s by using sols E and EW, led to smaller crys-tals (Fig. 7(a)), whereas the aqueous sol W led to the biggest(Fig. 7(c)); an intermediate situation is obtained from coat-ings deposited by using sol WD (1 : 2 ratios, and speed of2500 µm/s). In a previous work, we observed that HA coatingsdeposited by using aqueous sols showed the formation of thislayer after the immersion into a SBF solution [16]. These min-

Fig. 6. SEM micrographs of an HA coating deposited by using sol WD soaked 7 days in SBF (a); the same coating but higher magnification (b).

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Fig. 7. SEM micrographs of HA coating deposited soaked 7 days in SBF by using sol E (a); sol WD (b) and sol W (c).

eralized layers were also constituted by small crystallites whosesize decreases with the soaking time and the coating thickness.

The differences observed in the mineralized layer can be ex-plained by the dissolution and precipitation behavior that HAsol–gel coatings experiment when immersed in SBF. Theseprocesses are two of the main factors governing the bioactiv-ity of HA coatings [37]. Depending on parameters such as theviscosity of the sol or the withdrawal speed, the HA coatingsdeposited show different thickness and, consequently, differentceramic content. The quantity of HA deposited onto the sub-strate influences decisively on the crystal size of the mineralizedlayer, i.e., a higher content of ceramic provokes a rapid satu-ration of the medium followed by an immediate precipitationprocess characterized by the proliferation of numerous crystal-lization seeds, inducing mineral layers with small grain size.On the contrary, less thick coatings provoke a slower precipita-tion rate, generating fewer nuclei and, consequently, yielding alayer compound by bigger crystals.

The composition/structure of this new mineralized layershould be very similar to the HA sol–gel coating deposited,hence it is very difficult to distinguish it by XRD and FTIR.In this sense, a study by ED and TEM was performed scrap-ping the material from the surface of the substrate.

Fig. 8 shows the TEM micrographs with the correspond-ing ED patterns and EDS spectra of the two types of particlesscratched from the surface of the substrate after 7 days in SBF.The crystals corresponding to the sol–gel coating (Fig. 8(a))are very small (<25 nm), round-shaped and with a Ca/P mo-lar ratio of 1.7 ± 0.1, according to the EDS spectra. The onesformed into the SBF solution (Fig. 8(b)) are larger and show aneedle-like shape; the Ca/P molar ratio of this kind of crystalswas found at 1.4 ± 0.1, similar to the ratio observed in biolog-ical apatites [38] and other apatites formed in SBF [36]. In thesame way, both ED patterns show diffraction rings that can beindexed to the interplanar spacings of an apatite phase. In ad-dition, the ED diagram in Fig. 8(a) shows, as well, diffractionmaxima which are indicative of the higher crystallinity of sol–gel derived HA nanocrystals when compared to those obtainedin SBF.

Consequently, the in vitro bioactivity/dissolution test showsthat the apatite coatings deposited by sol–gel method are stablein the fluid, that is, there are coating remains after the immer-sion into the medium. This HA sol–gel coating reacts with theions present in the medium forming a new apatite layer in itssurface. This mineralization behavior has been observed in all

Fig. 8. TEM micrographs, ED patterns and EDS spectra corresponding to thecrystals formed on the sol–gel coating (a); new layer formed after 7 days inSBF (b).

the HA coatings independently of the precursor sol characteris-tics.

4. Conclusions

Nanocrystalline carbonatehydroxyapatite coatings ontometallic substrates have been prepared by a sol–gel techniquefrom precursors which differ in the ethanol/water content. Inall the cases a control of the aging time is necessary in orderto avoid a phase mixture or poor coating surfaces. All the solsform uniform and homogeneous films on substrate, increasingthe uniformity in presence of ethanol.

The HA coatings obtained show good adhesion values, about20 MPa and a bioactive behavior under in vitro conditions,inducing bone-like apatite formation on their surface when im-mersed in SBF.

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The results obtained in this work indicate that a compromiseamong the aging time of sol, coating uniformity, thickness androughness must be reached in order to make these precursorsgood for an industrial use. The short aging time for the sol aswell as a wider interval time to deposit coatings, would increasethe efficiency of the process, which is favorable for an indus-trial application. One option could be to use precursor-sol typeWD, that is, to add the ethanol after the hydrolysis/condensationprocess; in this way, lower aging times are necessary to obtainHA coatings, it allows to deposit HA layers in a wide time in-terval, the sol covers homogeneously the substrate and higherwithdrawal rates can be used, which allow to prepare thickercoatings.

Acknowledgements

Financial support of CICYT (Spain) through ResearchProject MAT2005-01486 is acknowledged. The authors thankDr. J. Llopis for the use of a spectrophotometer for thicknessmeasurements. The XRD, SEM, TEM and SFM measurementswere performed at C.A.I. Difracción de Rayos X and Micros-copia Electrónica, Universidad Complutense, respectively. Theauthors thank Biomet Spain Orthopaedics for providing the ti-tanium alloy.

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