98. 1. effect of various sterilization methods on the bio activity of lasere ablation...

Effect of various sterilization methods on the bioactivity of laserablation pseudowollastonite coating

F. A. Zuleta, P. Velasquez, P. N. De Aza

Instituto de Bioingenieria, Universidad Miguel Hernandez, Elche, Alicante, Spain

Received 2 November 2009; revised 8 March 2010; accepted 14 April 2010

Published online 24 June 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31667

Abstract: Sterilization is required for using any material or

device in contact with the human body. The aim of this work

was to investigate the effect of four sterilization methods

(steam autoclave, hydrogen peroxide plasma, ethylene oxide,

and gamma sterilization) on the surface chemistry and in

vitro bioactivity of pseudowollastonite (psW) coatings in tita-

nium alloys substrates. psW coatings in Ti-6Al-4V substrates

obtained by laser ablation technique were sterilized and

immersed in Kokubo’s simulated body fluid (SBF) up to 30

days. No changes in the chemical composition were noted af-

ter sterilization. However, a Ca/P-layer of different thickness,

identified as hydroxyapatite (HA) like was developed on all

the samples after soaking, although, the ethylene oxide steri-

lized samples present a nonhomogeneous and �55.9% thin-

ner HA-like layer. VC 2010 Wiley Periodicals, Inc. J Biomed Mater

Res Part B: Appl Biomater 94B: 399–405, 2010.

Key Words: bioceramics, sterilization, coating, bioactivity, in vitro

INTRODUCTION

A biomaterial is a substance that has been engineered to takea form which, alone or as part of a complex system, is used todirect, by control of interactions with components of livingsystems, the course of any therapeutic or diagnostic proce-dure, in human or veterinary medicine.1 Because biomaterialsare selected based on the combination of mechanical, physical,and biological properties, investigators have found the need toenhance biocompatibility. The essential requirement for an ar-tificial material to bond to living bone is believed to be theformation of a biologically active apatite-like layer on theirsurfaces in the body.2–5 Limited number of ceramic materialscalled bioactive materials such as bioglass,2 hydroxyapatite(HA),5 glass-ceramic A-W,6 diopside,7,8 and pseudowollastonite(psW)9,10 are generally known to possess the ability to formbone like apatite in the body environment.

Unfortunately, in most of the cases, mechanical strengthof these materials were not enough to use them as bulkimplants, and hence, their applications have been limited toless loaded portions. Nevertheless, their field of applicationcan be expanded if used as coatings on metallic implants. Inthis way, the benefits of the bioactive material are combinedtogether with the mechanical performance of metallicimplants. The most common example of this type of combi-nation is titanium coated with HA.11–13 Our past and cur-rent studies demonstrated the formation of a HA-like layeron the surface of psW ceramic ‘‘in vitro’’14,15 and‘‘in vivo.’’9,10,16,17 This finding is very significant, as it indi-cates that the psW can be physically and chemically inte-grated into the structure of living bone tissue, and therefore,could be suitable for repair or replacement of living bone.

Because sterilization is the final step in manufacturingany implant device, the effect of sterilization processes onthe modification of the first surface layers of these biomate-rial surfaces must be clearly understood and characterized.Furthermore, the relation between the physico–chemical fac-tors and the tissue response must also be investigated.Moreover, the requirement for a well-controlled surfacebefore implantation emphasizes these needs. Shabalovskayaet al. reported on the correlation between the surface chem-ical composition and some sterilization treatments.18,19 Asimilar study on commercially pure titanium also showedthe potential damaging effect of some sterilization treat-ments such as ethylene oxide and steam autoclaving on thein vitro biological response.20 In spite of a wide applicationof bioceramics in dentistry and medicine, surprisingly, thereare only a few publications devoted to the influence of thesterilization conditions on the chemical properties of biocer-amics.21–23

So, it appears to be both interesting and important tostudy the influence of different sterilization methods on thechemical properties of the bioceramics. The aim of thiswork was to study the effects of the most accessible sterili-zation techniques on the surface chemistry and in vitro bio-activity of psW coatings in titanium.

MATERIALS AND METHODS

The starting material for this study was psW ceramic syn-thesized by a solid stated reaction from a stoichiometricmixture of calcium carbonate and high purity washed Bel-gian sand (99.9 wt %). Details of psW preparation and

Correspondence to: P. N. De Aza; e-mail: [email protected]

Contract grant sponsor: CICYT; contract grant number: MAT2006-12749-C02-02

Contract grant sponsor: Generalitat Valenciana; contract grant number: ACOMP/2009/173

VC 2010 WILEY PERIODICALS, INC. 399

characterization can be found in previous publications.14–16

Pellets of synthesized psW were axially pressed in a press-ing die of 30 mm of diameter at 150 MPa. A 1 wt % of a so-lution of 0.11 wt % of a plasticizer Zusoplast 91/11 wasadded and further sintered at 1350�C for 2 h. The sintereddisk had a diameter of about 25 mm and a relative densityof about 96%. This disk was used as target for pulsed laserdeposition.

The coatings substrates were 11 cm2 of Ti-6Al-4V with athickness of 1.5 mm. All the samples were obtained by follow-ing the procedure already described in a previous work.13

This procedure combines the pulsed laser deposition and thelaser surface treatment. First, Ti-6Al-4V substrates werecoated with psW through pulsed laser deposition from a bulkpsW target. A pulsed Nd:YAG laser, delivering 10 ns pulses of70 mJ at a wavelength of 355 nm and a repetition rate of 10Hz, was used for laser ablation. The laser fluence on the psWtarget was 2.5 J/cm2. The substrates were placed at 4 cm infront of the target, and during deposition, the substrates wereat 550�C in a 10 Pa oxygen atmosphere. Each coating resultedfrom 36,000 laser pulses.

After deposition, the samples were treated in air with acontinuous wave Nd:YAG laser to improve the density of thecoatings and their adhesion to the substrate. The laserbeam, with a wavelength of 1064 nm and a power of 35 W,was focused on the coatings surface at 36 kW/cm2. Thelaser beam scanned their entire area at a speed of 250mm/s line by line with steps of 100 lm.

Twelve pellets of samples were processed under clinicalconditions by common sterilization process used in hospi-tals including steam autoclave, hydrogen peroxide plasma,ethylene oxide, and gamma sterilization irradiation steriliza-tions. Nonsterilized psW-titanium samples were used ascontrols

Steam autoclave (EVS 425.2.M)Sealed bags with the samples were sterilized in autoclaveaccording to the standard procedure DIN 58946 used inmedicine. A set of samples were sterilized at 121�C. Briefly,a single run of the steam sterilization procedure in auto-clave includes three stages: temperature increasing up to121�C during 15 min with simultaneous pressure increasingfrom 0 to 1.4 mbar, sterilization at 121�C during 15 min atpressure of 1.4 mbar, followed by fast (1–2 min) tempera-ture reduction to 25�C. Thus, the total time of a single runof the steam sterilization procedure is about 45 min.

Hydrogen peroxide plasma (Sterrad)Plasma sterilization treatment was carried out in a largevolume microwave plasma (LMPTM) reactor. A set of sam-ples were sterilized exposing to a 58% hydrogen peroxideplasma for 15 min. The gas was introduced into the cham-ber at an operating pressure of 500 MTorr, and plasma wasexcited with 400 W of microwave power.

Ethylene oxide (Steri Vac EOE-M.)The units were sterilized by exposing to a 100% ethyleneoxide atmosphere for 156 min at 55�C and a gas concentra-

tion of 900 mg/L. After sterilization, the samples are aer-ated with warm air flow (37�C) at atmospheric pressure for330 min to remove residual ethylene oxide, and stored inindicator bags and sealed.

Gamma sterilization (Rhodotron TT200)Gamma irradiation sterilization with a 60Co irradiator wasperformed by Ionmed S.A. (Cuenca, Spain). Randomizedsamples were packed and sealed in indicator bags andexposed to gamma irradiation in three cycles at doses of18.71 kGy, 19.17 kGy, and 18.86 kGy, resulting in a dose of56.34 kGy.

After being ultrasonically washed in acetone and rinsedin deionized water, specimens were soaked in 100 mL ofsimulated body fluid (SBF) in polyethylene bottles at36.5�C. The immersion period of the specimens in SBF was21 days. This period was chosen based on the results fromprevious in vitro experiments performed in the SBF.14,15,24

At periodic intervals, the pellets were removed from thefluid and were left to dry in air at room temperature.

Surface of the samples were characterized before and af-ter the sterilization treatments. The crystalline structure ofthe coatings was analyzed by X-ray diffractometry (XRD)and Raman spectroscopy in the range 100–1200 cm�1. Thesurface morphology and composition were investigated in ascanning electron microscope (SEM) using a Hitachi S-3500N, equipped with detectors of secondary and backscat-tered electrons and fitted with X-ray energy-dispersive spec-trometer (EDS). The coatings thickness was measured witha surface profilometer.

The structural changes of the psW coatings after immer-sion in SBF for various periods were first analyzed by thin-film X-ray diffraction (XRD), which enables analysis of up toa 1-lm thick layer, depending on the incident X-ray beamangle. The morphology of the surface and the cross sectionsof the specimens were examined by SEM and EDS. The crosssections studied in the SEM were polished to 1 lm finishusing diamond paste, gently cleaned in an ultrasonic bath,and carbon coated. X-ray elemental maps of Si, Ca, and P ofthe specimens were also obtained. The thickness of the pre-cipitation layer formed on the psW coatings surfaces wereevaluated from the SEM photographs of the cross section ofthe soaked psW coatings samples. Additional changes inionic concentration, using inductively couple plasma atomicemission spectroscopy (ICP), were determined.

The product of the surface reaction was subsequentlycharacterized using transmission electron microscopy (TEM)technique. Jeol Jem 2010 microscope was operated at 200keV, and 80 cm camera length condition was applied forselected area diffraction patterns (SAD). These specimenswere prepared by careful removal of the reaction layer fromthe specimen’s surface using razor blade, and dispersing thepowder on the surface of alcohol in Petri dish. The powderspecimens were then collected on carbon coated copperTEM grids of 200 mesh and carbon coated. Electron beamtransparent particles were chosen for TEM examination bySAD, high-magnification imaging, and EDS analysis.

400 ZULETA, VELASQUEZ, AND DE AZA STERILIZATION METHODS ON BIOACTIVITY PSEUDOWOLLASTONITE COATING

RESULTS

Figure 1(A) shows SEM photograph of the surface micro-structure of the coating after laser treatment. It can be seenthat the coating is characterized by a compact and roughsurface, with some partially melted particles. The analysis ofthe composition of the coatings by EDX gives the followingatomic ratios: Si/Ca ¼ 0.9 and O/Ca ¼ 2.9. These values arevery close to those of the psW: Si/Ca ¼ 1 and O/Ca ¼ 3.The microstructure of the steam autoclave method sterilizedpsW-coating sample as a representative of all the samplesstudied is shown in Figure 1(B). The picture is similar tothat of the control sample [Figure 1(A)] presenting asmooth surface with some partially melted particles.

The results obtained with XRD on the psW-coating sam-ples before and after sterilization are shown in Figure 2.The XRD of the psW powder used as a raw material for thecoating is also include for comparative purposes. From Fig-ure 2(A), it can be seen that the psW powder has high crys-tallinity and there are only peaks corresponding to psW(high-temperature form of wollastonite-2M). Some changescan be noted in Figure 2(B), where in addition to the sharp

peaks corresponding to psW, they appear those of the sub-strate. The results obtained from Figure 2(B,F) demonstratethat there are no differences before and after sterilization.The diffracted peaks can be unequivocally identified as psWplus some sharp peaks of the titanium substrate.

The results obtained with Raman spectroscopy on thepsW-coating samples before and after sterilization areshown in Figure 3. These results demonstrate that no signif-icant chemical changes happen when the samples are steri-lized. Like the results of XRD, no differences before and af-ter sterilization were observed for all the samplesinvestigated.

Figure 4 shows the SEM micrographs of the psW-coatingcontrol, the steam autoclave, and ethylene oxide sterilizedsamples after different soaking times into SBF. Steam, Ster-rad, and gamma samples present similar behavior than thepsW-coating control with a similar type of the precipitates.After 7 days of immersion, the material surface is coveredby a layer of small globular particles smaller than 5 lm indiameter, some of which reaches the size of 6 lm after 30days of soaking. After 1 week, these globular particles coverhomogeneously the whole surface of the material. The par-ticles morphology does no further change with soakingtime, although the density of the layer increases.

FIGURE 1. SEM micrographs of the (A) surface of the psW-coating

before sterilization (control sample). (B) Steam autoclave sterilized

sample as a representative of all the sterilized samples.

FIGURE 2. X-ray diffraction of (A) pseudowollastonite powder (B)

psW-coating before sterilization (control sample). (C) Steam auto-

clave, (D) Sterrad, (E) ethylene oxide, and (F) gamma sterilized sam-

ples. (*) ¼ Pseudowollastonite and (�) ¼ Ti-6Al-4V substrate.

FIGURE 3. Raman spectra of the (A) control sample (nonsterilized)

and (B) steam autoclave, (C) Sterrad, (D) EtO, and (E) gamma steri-

lized samples (All the peaks correspond to pseudowollastonite.).

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2010 VOL 94B, ISSUE 2 401

SEM micrographs of ethylene oxide sterilized samples,namely, EtO in short, after different soaking times into SBFare also shown in Figure 4. The behavior detected was simi-lar to that previously mentioned, although a differenceshould be pointed out. After soaking time of 7 days, isolatedaggregates of globular particles are detected on the surface,

partially covering the surface. EDX analysis shows that thesespherical particles are constituted by calcium and phospho-rous. After 14 days of immersion, these aggregates of par-ticles grow in size. This feature does not further change andthe surface is not fully covered by the spherical particles af-ter 30 days of immersion [Figure 4(B), EtO)]. After 30 days,

FIGURE 4. SEM images of the samples surfaces. Control sample (nonsterilized), steam autoclave, and EtO sterilized samples after immersion

into SBF during (A) 7 days and (B) 30 days.


the globular particles reach a size between 3 and 3.9 lm indiameter.

Figure 5 shows the relationship between the soakingtime and the thickness of the Ca/P-layer developed on thesurface of the psW-coating. In the control sample (nonsteri-lized), a significant thickness of Ca/P-layer has been formed,reaching a total thickness of �24.3 lm (61 lm) after 30days. The formation rate gradually slows from �2.3 lm perday, after 7 days of soaking, to 0.5 lm per day, after pro-longed soaking (4th week of soaking). That is to say, themeasurements reveal that the Ca/P-layer formation kineticdepends directly on the square root of soaking time. In thesterrrad, stem, and gamma sterilized samples, the morphol-ogy of the surface product was the same, although the reac-tion rate and the thickness of the layer after 30 days wereslight smaller. In Steam sterilized samples, the total finalthickness of the Ca/P-layer after 30 days of soaking is 23lm (61 lm), in Sterrad sterilized samples is 21 lm (61lm) and for Gamma method its 18.4 lm (61 lm).

The main difference was in the EtO sterilized samples.Here it is worth to point out that the layer detected in theEtO sterilized samples was not of uniform structure anddensity throughout the sample. In the areas which the layerwas present a very low-layer formation rate of about 1 lmper day, up to 9.4 lm (61 lm), after 7 days of soaking, wasdetected. The layer reached a total thickness of 12.1 lm(61 lm) within these zones.

Changes in the elemental ionic concentrations of SBF af-ter 30 days of immersion are shown in Table I. The compo-sition of the original SBF solution is also enclosed for com-parison. The chemical analysis of the SBF used in the studyagreed with the composition of the one reported in the lit-erature.25 It was found that calcium and silicon ion concen-trations in SBF slightly increased over the exposure timeindicating partial dissolution of the psW-coating. On theother hand, phosphorous ion concentration decreased moresignificantly because of the precipitation of Ca-P phase onthe surfaces of the psW-coating. Although the formation ofCa-P phase consumed some calcium ions, the calcium ionsdissolution from the psW-coating were more than those

consumed. This finding is in agreement with the fact thatthe EtO samples do not present the surface fully covered bythe globular particles after 30 days of immersion, as can beobserved in Figure 4((B), EtO).

TEM was used to examine the ultra structure of the sur-face product formed after the exposure of Steam autoclavepsW-coating sample to SBF for 30 days. High-resolution lat-tice image was performed on the Ca/P crystals formed atthe surface of steam autoclave psW-coating sample. Theindividual’s crystals were found to grow in close contactforming continuous phase. (002) Lattice plane image of0.344 nm spacing were well resolved in many areas [Figure6(A)]. The resolved lattices were found to be defect free,with no discontinuity or bending present. EDS analysis per-formed on the thin crystals confirmed the presence of cal-cium and phosphorous in the region [Figure 6(B)]. Themeasured lattice spacing (obtained from the image negative)matches well the values of 0.82 nm for HA reported in theliterature.26–31 The HA-like crystals which orientation variedfrom (100) did no fulfill the lattice imaging conditions.When the specimen was appropriately oriented, the SAD of-ten displayed (002) arcs corresponding to the 0.344 nm lat-tice spacing, indicating on the preferential orientation of theHA-like crystals in the layer. Figure 6(C) displays a goodmatch between the measured values of the lattice spacingsobtained from the SAD pattern [Figure 6(C)] and ASTM datafrom HA [Figure 6(D)].

DISCUSSION

The in vitro results carried out in SBF for the psW coatingsin titanium alloys substrates sterilized by steam autoclave,hydrogen peroxide plasma, ethylene oxide, and gamma steri-lization irradiation, suggest that all the sterilized materialshave in vitro bioactivity as shown by the formation of HA-like phase in SBF solutions and the mechanism of this bio-activity is in agreement with the mechanisms reported byother researcher.32–34

When immersed psW-coating in SBF solution, both dis-solution and HA-like precipitation occur on the surface onthe materials. At early stage, the dissolution of psW-coatinggenerally proceeds faster than precipitation. The psW-coat-ing dissolved little before the HA-like precipitation processbecame dominant. When the psW-coating gets in contactwith the SBF, a partial dissolution occurs, producing an ionicexchange of Ca2þ for 2Hþ within the material network, lead-ing to the formation of silanol groups on the surface of the

FIGURE 5. Ca/P-layer thickness change as a function of soaking time

into SBF: Control sample (nonsterilized) and steam autoclave, Sterrad,

EtO, and gamma sterilized samples.

TABLE I. Concentration of Caþþ, Si4þ, and HPO2�4 , in SBF

before and after Immersion During 30 Days of the Control

Sample (Nonsterilized) and Steam Autoclave, Sterrad, EtO,

and Gamma Sterilized Samples

Samples (mg/L) Caþþ Si4þ HPO2�4

SBF control solution 100.36 – 93Control pellet 104.23 8.64 63.98Steam autoclave 102.58 7.582 65.27Sterrad 106.39 7.24 74.37Gamma 101.73 8.45 77.97EtO 102.17 7.87 83.01



psW-coating. Later, there is a partial dissolution of amor-phous silica as SiO2�

3 . This fact enhances the formation ofcrystallization nuclei for the HA-like phase, which can beformed from the high concentration of Ca2þ and HPO2�

4

present in the media. Before immersion in SBF, the psW-coating had an average thickness of 18 lm (61 lm). Afterimmersion in SBF for 30 days, its thickness decreased, forall the sterilization methods, in average up to 12 lm (61lm), and the precipitated HA-like grew uniformly in averageto a thickness of 21.62 lm (61 lm) for the control and allsterilized samples except EtO, where the precipitation is nothomogeneous.

In the areas which the layer was detected, a low-layerformation rate of about 1 lm per day was measured. After7 days of soaking, the layer reached 9.4 6 1 lm attaining,in these areas, a total thickness of 12.1 lm (61 lm). Thismean that, in the EtO sterilized samples, the total Ca/P-layerformed, after 30 days of immersion was about 55.9% thin-ner than in the other sterilized samples. This is in agree-ment with the previously mentioned results obtained bySEM during the surface studies [Figure 4(B), EtO] where theEtO sterilized samples do not present, after 30 days ofimmersion, the surface fully covered by the Ca/P globularparticles. On the other hand, the ICP-AES results of phos-phorous ion concentration in SBF (Table I) shows that theminor depletion in P corresponds to the EtO sterilized sam-ples. One of the reasons for this difference, in the EtO steri-lized samples, is due to some residual ethylene gas that pos-sibly is present on the surface of the material after

sterilization.35–39 As it was reported elsewhere,35–39 thereare problems with the toxicity and carcinogenicity of the re-sidual ethylene gas in materials. The residual ethylene gasshould also affect the nucleation and crystal growth of HA-like. Our next step will be to implant the sterilized materialsto study the tissue responds.

On the other hand, the aspect of all sterilized ceramicsurfaces after 30 days of soaking is comparable with thatshown by other silica-based materials. Generally, the HA-likeformation occurs in two stages: a previous formation ofglobular particles followed by the apparition of aggregates,which after 10–15 days of soaking are being to be indistin-guishable.24,32,33,39 The comparison of the results obtainedfor all sterilized materials points to a disperse nucleation ofan HA-like phase on EtO sample. Globular aggregates can bedetected from the 1st week of soaking and are heterogene-ously distributed over EtO sample surface. Instead, the othersterilized methods samples seems to nucleate homogene-ously so that the observed layer is formed from a largernumber of crystallization nuclei homogeneously distributedsince the 1st week of immersion. The individual growth ofthese leads to the HA-like layer formation. The comparisonof the results obtained for all sterilized materials points alsoto a disperse nucleation of an HA-like phase on EtO sample.Globular aggregates can be detected from the 1st week ofsoaking and are heterogeneously distributed over EtO sam-ple surface. Instead, the other sterilized methods samplesseems to nucleate homogeneously so that the observedlayer is formed from a larger number of crystallizationnuclei homogeneously distributed since the 1st week ofimmersion. The individual growth of these leads to the HA-like layer formation.

CONCLUSIONS

The influence of four sterilization methods (steam autoclave,hydrogen peroxide plasma, ethylene oxide, and gamma steri-lization) on the surface chemistry and the in vitro bioactiv-ity of psW coatings in Ti-6Al-4V substrates wereinvestigated.

The XRD and Raman spectroscopy results appeared basi-cally similar; it has been shown that there was no signifi-cant modification of the psW-coating structure in the sur-face of the samples by the four sterilization methods used.

SEM observation, after the SBF soaking, showed essen-tially identical surface morphology regardless of the sterili-zation by ethylene oxide, where the HA-like layer does notfully cover the surface of the samples after 30 days ofimmersion. One of the reasons for this difference is due tosome residual ethylene gas that possibly is present on thesurface of the EtO sterilized samples after sterilization. Thisresidual ethylene gas should affect the nucleation and crys-tal growth of HA-like.

The sterilized materials have in vitro bioactivity with theformation of an HA-like layer on the surface of all the sam-ples, although the ethylene oxide sterilized samples presenta �55.9% thinner and nonhomogeneous HA-like depositionthroughout the samples. Therefore, we recommended notusing the EtO sterilized method for this type of materials.

FIGURE 6. (A) HRTEM micrograph of the HA-like phase formed during

exposure to SBF after 30 days (steam autoclave sterilized sample), (B)

EDX analysis, (C) SAD pattern of the phase, and (D) lattice spacing of

the HA-like phase, taken from SAD of (C) and ASTM data.


ACKNOWLEDGMENTS

The authors thank Dr. Luna from Ionmed SA (Cuenca, Spain)for the gamma sterilization and Dr. F. A. Lopez Prats for thesteam autoclave, hydrogen peroxide plasma, and ethylene ox-ide sterilizations.

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98. 1. effect of various sterilization methods on the bio activity of lasere ablation...

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