Antifungal activity of Ag:hydroxyapatite thin films synthesized bypulsed laser deposition on Ti and...

8/13/2019 Antifungal activity of Ag:hydroxyapatite thin films synthesized bypulsed laser deposition on Ti and Ti modified by

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Applied Surface Science 293 (2014) 3745

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Antifungal activity ofAg:hydroxyapatite thin films synthesized bypulsed laser deposition on Ti and Ti modified by TiO2nanotubessubstrates

S. Erakovic a, A.Jankovic a, C. Ristoscub, L. Dutab, N. Serbanb, A. Visanb, I.N. Mihailescub,,G.E. Stanc, M. Socol c, O. Iordached, I. Dumitrescud, C.R. Luculescub, Dj.Janackovica,V. Miskovic-Stankovic a

a Faculty of Technology andMetallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbiab National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, 077125 Magurele-Ilfov, Romaniac National Institute of Materials Physics, 105 bis Atomistilor Street, 077125 Magurele-Ilfov, Romaniad National Institute for Textile and Leather, 16 Lucretiu Patrascanu, 030508Bucharest, Romania

a r t i c l e i n f o

Article history:

Received14May2013

Received in revised form

28November2013

Accepted7 December2013

Available online 21 December 2013

Keywords:

Antifungalactivity

Ag doped thin films

TiO2nanotubes

HydroxyapatitePulsed laser deposition

a b s t r a c t

Hydroxyapatite (HA) is a widely used biomaterial for implant thin films, largely recognized for its excel-

lent capability to chemically bond to hard tissue inducing the osteogenesis without immune responsefrom human tissues. Nowadays, intense research efforts are focused on development of antimicrobialHA doped thin films. In particular, HA doped with Ag (Ag:HA) is expected to inhibit the attachment

of microbes and contamination ofmetallic implant surface. We herewith report on nano-sized HA andAg:HA thin films synthesized by pulsed laser deposition on pure Ti and Ti modified with 100nm diam-

eter TiO2 nanotubes (fabricated by anodization ofTi plates) substrates. The HA-based thin films werecharacterizedby SEM, AFM, EDS, FTIR, andXRD. The cytotoxic activitywas tested withHEp2 cells against

controls. The antifungal efficiency of the deposited layers was tested against the Candida albicans andAspergillus nigerstrains. The Ti substrates modified with TiO2nanotubes covered with Ag:HA thin films

showed the highest antifungal activity. 2013 Elsevier B.V. All rights reserved.

1. Introduction

Hydroxyapatite [HA,Ca10(PO4)6(OH)2] bioceramic thinlayersascoatings for metallic medical implants, which were demonstratedto promote the formation of bone-like apatite on their surfaceimproving the implant fixation andpromoting the bone in-growth

[15], still remain a research topic of great interest.As a metal substrate for regenerative medicine applications,

such as orthopedic or dental implants, titanium (Ti) is widelypreferred due to its desirable properties, such as suitable elastic

modulus and mass density, good mechanical strength, corrosionresistance, biocompatibility and low toxicity [2,68]. However, Tiexhibits a rather poor bioactivity and in order to establish a directchemical bonding between the implant andthehost bone tissue its

adequate surface modificationwas suggested using various routes[9]. Oneapproach to enhance thebioactivity and improve theboneresponse to implant surface isbydepositing bioactiveHA thin filmson Ti implant surface.

Correspondingauthor. Tel.: +40 21 4574491; fax: +4021 4574243.

E-mail address: [email protected](I.N.Mihailescu).

Recently, nanotubular titanium dioxide (TiO2) layers haveattracted an increased attention in comparison to pure Ti due to

their ability to enhance the bonding strength between HA coat-ing and metallic substrate [1012]. The nanotubes fabrication byelectrochemical anodic oxidation of pure Ti in fluoride-containingelectrolytes provides strongly adherent layers which are used for

variousapplications,basedupon theirsemiconductiveandbiocom-patible nature [12].

A challenging task in biomedicine is the prevention of micro-bial infectionswhichdetermine the looseningof implants from the

bone [8,13]. A promising strategy to prevent the initial microbialadhesion and colonization of biofilms is via introducing antimi-crobial bioactive thin films onto implants surface. Some previousstudieshave shown that Ag+ ions have a broad spectrumof antimi-

crobial and antifungal properties [14] while maintaining a lowcytotoxicity [4]. The Ag+ ions are able to penetrate the microbialcellwall and bind toDNA, and thus interferingwith the replicationprocess [1517]. Currently, special interest is focused on thedevel-

opment of HA coatings doped with silver to minimize microbesadhesion [18].

There are various methods to deposit ceramic thin films onmetalsurfaces, suchasplasma spraying [19],magnetronsputtering

0169-4332/$ see front matter 2013 Elsevier B.V. All rights reserved.

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38 S. Erakovic et al. / AppliedSurface Science293 (2014) 3745

[20], pulsed laserdeposition [1,21], solgel [22], electrochemical or

electrophoretic deposition [2325]. Pulsed laser deposition (PLD)is a technique that has proved efficient in the fabrication of cal-cium phosphatefilms onmetallic substrates with excellentcoatingattachment [1]. Moreover, by a proper choice of the ablation and

deposition parameters, it is possible to control the stoichiometryandcrystallinityof a wide range of complex materialsdepositedatroom temperature (RT) [1,2].

We herewith report on the physicalchemical characterization

of HA and Ag doped HA [Ca9.95Ag0.05(PO4)6(OH)2] thin films, withAg content of 0.530.1wt.%, synthesized by PLD onpure Ti and Timodified by TiO2 nanotubes substrates. The antifungal efficiencyof the novel Ag:HA/TiO2 nanotubes structures was tested against

twopathogenic,largely spread, fungal strains:Candidaalbicansand

Aspergillus niger. The specific aim of this manuscript is the iden-tification of the most suitable deposition substrates and optimalprocessing conditions.

2. Materials and methods

2.1. Preparation of Ti modified by TiO2nanotubes

Ti plates of 20mm10mm0.25mm (99.7% purity, SigmaAldrich) were used as substrates for the growth of nanotubes tita-nium oxide film. The substrates were degreased in acetone andethanol in an ultrasonic bath for 30min and rinsed with deion-ized water. Theanodization wasperformedunder constantmixing

in a two-electrode cell. The Ti plate was the working electrodeand a Pt plate was used as a counter electrode at a distance of 15mm in 0.4wt.% HF solution. The experiments were carried outby PEQLAB Power Supply EV231 and performed potentiostatically

(20V for 30min) at RT. After anodization, the samples were rinsedwith deionized water and air dried at RT. Prior to PLD experi-ments, the anodized Ti plates were thermally treated by using aULVAC-RIKO MILA-5000 instrument at 450C for 1h, in air with a

heating/cooling rate of 30 C/min.

2.2. PLD experiment

PLD targets (20mm diameter) were prepared by pressing HA

and Ag:HA (0.530.1wt.%) nanopowders at 3MPa and sinteringat 650 C for 6h. The heating/cooling ratewas set at 25/15 C/min.Ouroption forAg dopingconcentrationwasbasedupon the resultswe reported in Ref. [21]. As previously reported in detail [21],

we cultivated mesenchymal stem cells on compositional libraryof Ag and HA layers. It was shown that an Ag content of up to0.6wt.% into HA deposited by PLD could be considered nontoxicfor cells [21]. Accordingly,we have selected an Ag concentration of

0.530.1wt.% as the best compromise between thehighest possi-ble antimicrobial activity and absence of toxic reaction regarding

cell removal or apoptosis, in the case of PLD films.PLD was performed inside a stainless steel deposition cham-

ber. The deposition of the HA and Ag:HA thin filmswas conductedby ablation of respective targets with a KrF* excimer laser source(=248nm, FWHM 25ns). The repetition rate was of 10Hz and

the applied fluencewas set at 4.5 J/cm2 (with a corresponding laserenergy of 435mJ). For the deposition of each film, 15,000 subse-quent laser pulses have been applied. During the multipulse laserirradiation process, the targetswere rotated with 0.4Hz and trans-

latedalong two orthogonal axes to avoid drilling and to ensure thedeposition of an uniform film.

The filmswere deposited at 50Pa in water vapors flux on Ti oranodized Ti substrates that were heated at 500C. For some anal-

yses, twin samples were deposited on 111 single-crystalline Si

wafers. A target-to-substrate distance of 50mm was used in all

experiments. In order to explore the effect of the post-deposition

treatment in restoring the stoichiometry and improving the crys-tallinity of the thin films, half of the samples were submitted toa post-deposition thermal treatement for 6h at 500 C in a watervapors enriched atmosphere. The heating/cooling rate in this case

was20/10 C/min.Dependingon thetop apatite layerandsubstratenature the samples will be further denoted asHA/Ti (HA depositedon Ti substrate), HA/nTiO2/Ti (HA deposited on Ti modified by TiO2nanotubes), Ag:HA/Ti (silver doped HA deposited on Ti substrate)

and Ag:HA/nTiO2/Ti (silver doped HA deposited on Ti modified byTiO2nanotubes), respectively.

2.3. Characterization of deposited films

2.3.1. Physicalchemical analysesThesurfacemorphology of thedepositedfilmswasinvestigated

byfield emissionscanningelectronmicroscopy (FE-SEM)with a FEI

Inspect S electron microscope. The investigations were performedat 20kV acceleration voltage, in high vacuum, under secondaryelectrons acquisition mode. The samples were coated with a thinAu film in order to prevent electrical charging. Cross-section SEM

images were recorded on HA and Ag:HA thin films deposited onSi wafers in order to evaluate the thickness. Compositional energy

dispersive spectroscopy(EDS) analyseswere performedwith a SiLitypedetector (model EDAX Inc.), operated at 20kV. The EDS analy-

ses were conducted in duplicate on film regions having areas of250m250m. Both sets of experiments lead to comparableresults and for that reason, onlyresults fromone of the twoquanti-tativeanalysesarepresentedin thepaper.The thinfilms roughness

wasexaminedby atomic forcemicroscope (AFM),usinga NanonicsMultiview4000apparatus in tapping mode with a feedback phase.

Fourier transform infrared (FTIR) spectroscopy study wasconducted with a Perkin Elmer BX Spectrum spectrometer, in

attenuated total reflection mode, using a Pike-MIRacle diamondhead of 1.8mm diameter. The spectra were recorded in the rangeof 4000550cm1, with a resolution of 4cm1 and a total of 50 scans/experiment.

Identification of crystalline phases in the PLD thin films wascarried out by X-ray diffraction (XRD) in symmetric geometry,usingaBrukerD8 advancediffractometer,withCuK (=1.5418A)radiation. The diffractometer is equipped with a high efficiency

one-dimensional detector (Lynx Eye type) operated in integrationmode. The scattered intensitywasscanned in the 2range 2060,with a step size of 0.04, and 10s/step.

2.3.2. Cytotoxicity assayA HEp2 cell line (ATCC CCL-23TM) was used to assess the

cytocompatibility of the PLD films in terms of cellular adhesion,viability and proliferation. Dulbeccos Modified Eagle Medium:

Nutrient Mixture F-12 (DMEM:F12) (Sigma, USA) supplementedwith10% fetal bovineserum(FBS)wasused ascell culturemedium.

Prior to biological testing each material (glass control, Ti-control,nTiO2/Ti-control, and Ag:HA/nTiO2/Ti) was sterilized for 1h at

180 C in dry atmosphere. Cells were harvested from the sub-strate using a trypsin:EDTA treatment and brought to a density of1.5105 cells/mL in DMEM:F12 medium supplemented with 10%

FBS. Further, thecell suspensionwasplaced onbiomaterialsurfacesandallowedtogrowfor48hat37 Cina5%CO2 humidatmosphere.

The adherence of the cells to materials and cells viability wereevaluated using fluorescein diacetate (10g/mL) and propidium

iodide(10g/mL) stain,and their fluorescencewasquantifiedwithan Observer.D1 Carl Zeiss microscope.

After 48h the cells were harvested from the sample sur-faces, washed in a cold phosphate buffered saline (PBS) solution

(pH=7.5), and then fixed overnight in 70% ethanol at 20 C. The

samples were washed with PBS, treated with RNAse A (1mg/mL)


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S. Erakovic et al. / AppliedSurface Science293 (2014) 3745 39

and labeled with propidium iodide (100g/mL) at 37 C for 1h.

Then, theDNA content of cells was quantified on a Beckman Coul-ter EPICS XLflow cytometer and analyzed by FlowJo8.8.6 software(Ashland, OR, USA).

2.3.3. Assessment of antifungal activityThe antifungal efficiency of the deposited thin filmswas tested

according to ASTM E2180-07 (2012) standard. This method canbe used to evaluate effectiveness of incorporated/bound antimi-crobials in hydrophobic materials. The aqueous based microbialinoculum remains in close, uniform contact in a pseudo-biofilm

state with investigated material. The percent reduction in the sur-viving populations of microbial cells at 24h vs. those recoveredfrom the control was determined.

The colony plate count method was used for quantifying the

antifungal effect. The test was applied to two sets of specimens:

C. albicans (ATTC 10231) and A. niger (IMI 45551). C. albicans isthe most common fungus that causes the infection by adhesiononto implant surfaces leading to biofilms formation. It is therefore

considered a dangerous pathogenicmicroorganism [26].A. nigerisusually causing the formation of so-calledblackmold. Thespores

are especially dangerous when inhaled. Moreover, these fungalcolonies can be found in ventilation ducts in health care facilities

[27].The solvent for the inoculumwas an Agar slurrywhich reduces

the surface tension and allows for the formation of a pseudo-biofilm, providing an even contact with the test surface. Decimal

dilutions were prepared in sterile physiological saline buffer(0.877% (w/v), pH 7.2) until a final dilution of 103 and a concen-tration of 3.9105 CFU/mL for C. albicans and of 7.4105 CFU/mLforA. niger.

The samples were incubated along with the inoculum for 24hat a temperature of 37C for C. albicans, and 29 C for A. niger.After incubation, the samples were washed with sterile physio-logical saline buffer and the cells were passed on Petri plates with

Sabouraud,Dextrose-Agargrowthmediafor C.albicansandCzapek-Doxmedia, fortheA. niger. Next, thecolonieswerecounted, andtheresults were expressed as percentage reduction rate. All sampleswere tested in triplicate.

3. Results and discussion

3.1. FE-SEM, AFM and EDS

3.1.1. Morphology of Ti plates modified by TiO2nanotubesAs known [12], TiO2 nanotubes fabricated by anodization

method consist of patterned, strongly adherent nanostructures

with an increased surface area compared to planar Ti. Such

nanoscale morphology plays a pivotal role as the bone in-growthtakes place preferentially in pores.

Recently, it was demonstrated for a wide variety of materials

that nanostructured surfaces are enhancing the osteoblast cellu-lar functions, improving the osteointegration response [2830].Superficial nanostructuring or nanoroughing can increase boththe implant surface area and surface energy leading to a greater

interaction of specific cell adhesion proteins [29]. Moreover, ananoscale topography could promote unique energetic features,due to altered electron delocalizations or surface defects, actingin favor of a rapid osteointegration [31,32].

Asvisible from theFE-SEMmicrographs(Fig.1), the anodizationprotocol described in Section 2.1 lead to theuniform coating of theTi substrate surface by a thin layer of TiO2 nanotubes having an

inner diameter of80nm and outer diameter of100nm.

Fig. 1. FE-SEM micrographs of Ti modified by TiO2 nanotubes obtained after

anodizationat 20V for30minin 0.4wt.%HF solution:generalview(a) and detailed

view (b).

3.1.2. Morphology, structure and composition of HA and Ag:HA

thin filmsThe surface morphology, structure, composition and coating

thickness of HA and Ag:HA thin films deposited on a silicon sub-strate were investigated by top-view FE-SEM (Fig. 2), cross-viewFE-SEM, AFM and EDS (Table 1).

Table 1

EDSelemental composition of thePLD depositedHA and Ag:HAthin films.

Element Intensity Concentration

(at.%)

Intensity Concentration

(at.%)

HA Ag:HA

C K 0.0102 10.3 0.0151 9.18

O K 0.0501 52.13 0.1128 51.98

Si K 0.0009 0.09

P K 0.1587 14.53 0.1889 15.63

Ca K 0.3708 23.31 0.3474 21.71

Ag L 0.0077 0.22

Ca/P 1.60 1.39


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Fig.2. Top-viewFE-SEMmicrographsoftheHA(a)andAg:HA(b)coatingsdeposited

byPLD onSi 111mirror polishedwafers.

Well-distinguishable spherical particulates 1501500nm in

sizewere observed for bothHA and Ag:HA films (Fig. 2). The moreflattened particles resulted due to the energetic impact with thesubstrate.

According to current PLD physics models [1,33], such particu-

lates are either expulsed directly from target by phase explosion,plasma recoil and surface instabilities or are forming by coales-cence of particles due to intense collisions during the transit fromtarget to substrate. A significantly higher density of particulates

was observed for the Ag:HA films (Fig. 2b).At the same laser fluence, if assuming a lower melting point for

the Ag:HA (less crystalline than the pure HA, aswill be shown fur-ther),the increasednumberof particulates foundontheAg:HAfilm

surface can be the result of the phase explosion process (explosiveboiling) due to the surface superheating, at temperature approa-ching the thermodynamic critical point.

The abundant presence of these particulates leads to the strong

increase of surface roughness, and can result in a superior in situanchorageof the implant, thuspreventingmicro-movementsof themedical device and providing the necessary initial stability untilbonewill grow and attach to the implant surface.

Based upon the cross-section micrographs, the thickness ofthe two films was estimated at 1.440.1m (for HA) and

1.640.1m (for Ag:HA), respectively.Fromaqualitativepoint ofview, theEDS spectra of theHAbased

films (datanotshown) indicatedthepresenceof typical apatiteele-ments only (Ca, P, O, C), along with the signal originating from theSi substrate. In the case of Ag:HA coating, except the characteris-

tic HAelements,Ag presencewas also emphasized. The absence ofother cations asserts the films purity.

The EDS quantitative data were collected in Table 1. A slightdecrease of Ca/P atomic ratio in the pure HA films with respect

to the theoretical HA stoichiometry (Ca/P =1.67), can be noticed.However, because the renownlimitedaccuracy of theEDSanalysistechnique, one should treat such quantitative results with caution.The decrease of the Ca/P atomic ratio from 1.60 in HA film to 1.39

in Ag:HA film, pleads in favor of the substitution of a part of Ca

ions by silver ions. We note that the Ca/P atomic ratio for the HA

pure or doped films synthesized by PLD is dependent on substrate

temperature and incident laser fluence [34].Next, the surface morphology of Ag:HA films was investigated

in higher detail by AFM. High resolution AFM images (not shownhere) evidenced that the films consisted in fact of quite small and

tightely packed spheroidal grains. The thin Ag:HA films exhibitedquite similar morphologies irrespective of the type of substrateused. The absolute roughness (Ra) was of28nm for Ag:HA/Ti andof29nm for Ag:HA/nTiO

2/Ti structures. It is therefore resulting

that the substrate morphology has a reduced influence on coat-ing roughness. This is to be expected in case of thick films (inour case of 1.5m). When increasing the thickness, the grow-ing film tendency to mimic the substrate relief is reduced, so that

if the thickness becomes large enough, the film morphology ismainlydetermined by the physical/chemical depositionprocesses.At nanometric level, the roughness is mainly determined by thecontribution of the smaller droplets, from which the entire film

envelope (matrix, andembedded larger droplets) is composed.Wementionthat beforedeposition,the roughnessof Ti andTi modifiedwith TiO2 nanotubes substrates was of 7 and 20nm, respectively.According to Narayanan et al. [35], a low scale HA coating rough-

ness candecreasethecontact anglesandcanaidingoodattachmentof osteoblasts to metallic substrates. It follows that the deposited

Ag:HA thin films exhibit a convenient nanostructuring for boneimplants.

3.2. FTIR

Fig. 3 displays the FTIR spectra recorded in case of HA (a,c) and

Ag:HA (b,d) films deposited on Ti (a,b) and Ti modified by TiO2nanotubes (c,d), before and after the post-deposition heat treat-ment.

All IR spectra are dominated by the vibration effects of the var-

ious Qn phosphate species, which cover the entire 1400400cm1

wave numbers region [36]. The threefold degenerate 3stretchingof phosphate stands forthe typical prominentvibrationband ofHAand consists of at least three sub-modes [37].

In the case ofHA/Ti and Ag:HA/Ti as-deposited samples (Fig. 3aand b), the band centered at 938937 cm1 corresponds to the 1non-degenerated symmetric stretchingof phosphategroups,whilethe bands positioned at 964963, 10291021, 10881087cm1

belong to the degenerated 3 asymmetric stretching vibrations ofphosphategroups[36,38]. Thepeaksat561558and599598cm1

are due to the degenerated 4 asymmetric bending vibration ofphosphategroup. The3and 4(PO4)

3 vibrationbandsareconsid-

ered the IRfingerprint of a HA structure. In the caseofHA/nTiO2/TiandAg:HA/nTiO2/Ti samples, similar FTIRenvelopeswererecorded(Fig. 3c and d), displaying the same vibration bands. To the dif-ference of films deposited on TiO2 nanotubes arrays, for the films

deposited on pure Ti it was possible to emphasize the symmetricstretchingvibrations of PPgroups inQ3, Q2 and Q1 units, asa faint

band centered at800cm1. The low intensity band positioned inthe rangeof 875870cm1, for all as-depositedfilms,is the indica-

tive ofHPO42 impurity ions presence.Thebroadandlowintensity

band situated in the range of 16001300cm1 can be attributed tothe 3antisymmetric stretching vibrations of carbonate functional

groups. This can be caused by the possible sample contaminationduringhandling.The undefinednoisyaspectof thisregion suggeststhat the presence of carbonate groups on both sites [A-type sub-stitution refers to the (CO3)

2 in the OH sites, whilst the B-type

substitution is due to (CO3)2 replacing (PO4)

3 ions without anadjacent OH ion [37]]. The 4and 1vibrationmodesof carbonatecanbe rarelydetectedbecauseof their veryweak intensities,whilst

2 bendingband, generallysituatedat875cm1,withanintensity

about one fifth that of3 band, is obscured in our case by the acid

phosphateband[38].Moreover,one caninfer that theas-deposited


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Fig. 3. FTIR spectra of HA (a) and Ag:HA (b) films deposited on Ti substrates, HA(c) and Ag:HA (d) films deposited on Ti modified by TiO2 nanotubes substrates, before andafter the thermal treatment.

films are strongly dehydrated, as neither structural (deformation

L or stretching S) nor absorbed water (bending and stretching)vibration modes have been observed on theentire 4000550cm1

range. It is known that the atomic disorder in the apatite latticeimpedes the incorporation of OH ions [39].

The regular shifts of the bands in the case of Ag:HA films sug-gest that the occurrence of a series of short-range order structuralmodifications. The broadening of vibration bands, emphasized forthe all the as-deposited films, can be associated with a low local

symmetry andhigh atomic disorder.Afterthepost-depositionheat-treatment, onenotices a remark-

able modification of spectra profile. The vibration bands becamesharper, and the peaks are better resolved. The splitting of the 4bending and3stretchingmodes of phosphatedenotean improve-ment of the crystalline status of the HA films. This phenomenon

manifestsmore radically in case of films deposited on bare Ti sub-strate. The shifting of the absorption bands after annealing could

be explainedby amodification of the crystalline/amorphous phaseratio. Thephosphatebandsshiftedcloser to thepositionscharacter-istic toaHAstandardstructure, thus pointing toanimprovement ofthetetrahedralstructureofthephosphateions.Thenewbandpeak-

ing at630cm1, originating from the libration mode of hydroxylgroup, highlighted the presence of structurally bound OH in theapatitic films. This is a supplementary proof of theefficiency of thepost-deposition heat-treatment in promoting the reconstitution of

HA in termsof compositionandstructure. Because theHPO42 ions

are known tobe lost in the160240C temperature range [40], thelow intensity sharp peak arising at 877876cm1, for the heat-treated films, can be solely associated to the 2 bending vibration

of carbonate. This assumption is backed by the correlated rise and

evolution of the bands due to 3 stretching vibration of (CO3)2

groups 1415cm1 (Fig. 3a and b vs. c and d). The positions of thecarbonatebands indicate the predominance B-type substitution in

thecrystal lattice ofHA [38]. TheB-typeis thepreferential substitu-tion in the human boneand isknown tohavebetter bioactivity andosteoinductivity [41]. The carbonatation could arise from the sig-naled contamination of the as-deposited samples, but more likely

is potentiated by theeasiness of substitution of (PO4)3 by (CO3)

2

ions during the post-deposition thermal processing.

3.3. XRD

The XRD analyses evidenced the hexagonal HA (space group

P63/m; ICDD file: 00-009-0432) as the predominant crystallinephase in all films (Fig. 4). The most intensive peaks of the pat-

terns were the Ti peaks of the substrate (ICDD file: 00-044-1294).The intensity scales of thegraphicalrepresentationsweretherefore

chosen to emphasize the lower intensity lines originating from thedeposited films.

In case of as-deposited films, the broad and shallow HA peaks,

are indicativeof thepoor crystallizationunder the used depositioncondition. All the as-deposited PLD films contained a significantamorphous component, as revealed by the pronounced halo cen-tered at 231. Exceptionally, theXRD patterns of theHA/Ti and

Ag:HA/Ti as-depositedfilms revealed thepresenceof a supplemen-tarypeak,positioned at236.2,which canbebestassociatedtoatitanium sub-oxide phase (TiO, ICDD file: 01-086-2352), as super-ficial product of the Ti substrate fabrication. Indeed, the bare Ti

substrate diffractogram (black line bottom ofFig. 4a), showed

besides this 11 1 reflection, other TiO reflection as well, 200 at


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Fig. 5. Fluorescencemicroscopy images ofHEp2 cellsgrownondifferent samples:glass control (a);Ti substrate (b);nTiO2/Ti substrate (c); andAg:HA/nTiO2/Ti structure(d).

Table 2

Mean crystallite sizes estimated from theFWHMof the(0 02) and (30 0) crystal planesreflections,by applyingthe Scherrer formula.

Sample type Space group D00 2 (nm) D3 00(nm) D002/D3 00

HAPLDtarget

Hexagonal, P63/m (176)

51.8 17.2 3.01

Ag:HA PLD target 32.5 10.6 3.07

HA/Ti heat-treated 74.2 50.3 1.47

Ag:HA/Ti heat-treated 62.3 35.8 1.74HA/nTiO2/Ti heat-treated 42.6 37.0 1.15

Ag:HA/nTiO2/Ti heat-treated 54.1 49.3 1.10

The mean crystallite size, approximated on the basis of theFWHMof the (00 2) and (30 0) crystal planes reflections by apply-ing the Scherrer formula [45] (Table 2), fully supports these

hypotheses. The lineswidthwascorrectedfor instrumentalbroad-ening using a CeO2 laboratory reference. The peaks broadeningdue to the film inner strainwas not considered. The crystallite size

determined from the 002 line breadth stands for the dimensionparallel to the c-axis, while the crystallite size determined fromthe 300 line breadth corresponds to the dimension projected inthe basal plane.

The crystallites growth was anisotropic in shape, being appar-ently influenced by the substrate nature, their average sizedecreasing in the presence of the TiO2 nanotubes arrays. Rathersimilar D002/D300 ratios were obtained when using the same

type of substrate, irrespective of film type (pure or doped). Theanisotropy of the apatitic films decreases when deposited on TiO2nanotubes (Table 2).

As compared to theparent targets, theshape factor (D002/D300)

is reduced (the crystallites becoming less oblate): two times incase of films deposited on bare Ti, and three times in case of filmsdeposited on Ti modified by TiO2nanotubes (Table 2).

On the basis of these results, one may expect a substantial

improvementof bioactivebehavior in case ofAg:HA/nTiO2/Tiheat-treated sample as stimulated by their structural development.These depositions were therefore selected for biological assays.

3.4. Biological assays

Our studies of cytotoxicity showed that the tested biomateri-

als do not influence the cellular adhesion, viability, morphologyand proliferation rate. Their development onto the testedmaterialsurfaces is similar to the glass control (Fig. 5).

Through the DNA quantification by flow cytometry the cellsare divided in three categories: G1 (normal cells), S (cells thathave started DNA synthesis in order to divide) and G2 (cellsthat have terminated DNA replication and will undergo mitosis).

The proliferation index (Ip) is currently defined as the per-cent of cells that have started and finished the DNA replication[Ip = (S+G2)100/(G1+ S+G2)]. As seen in Table 3, there are norelevantdifferencesregardingproliferationbetweenthetestedbio-

materials and glass control.

Table 3

Cellular cyclephasesofHEp2 cellsgrownon the investigatedbiomaterialsand glass

control.

Sample G1 (%) S (%) G2 (%) Ip(%)

Glass control 70.7 20.18 9.12 29.30

Ti 72.09 19.00 8.91 27.91

nTiO2/Ti 70.87 20.22 8.91 29.13Ag:HA/nTiO2/Ti 70.21 20.51 9.28 29.79


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Fig. 6. Comparativephotographsof fungal populations(Candida albicans aandb;Aspergilus niger c andd), 24h after incubation, onthe heat-treatedAg:HA/nTiO2/Tifilms

(b and d) and standard control samples (a andc).

Following the results of physicalchemical investigations andcytotoxicity tests, we selected for antifungal testing the heat-treatedAg:HA thin films deposited onmodified by TiO2nanotubessubstrates. The obtained results in case ofC. albicans and A. nigerstrains are given in Fig. 6 and summarized in Table 4 againstcontrol.

From the results presented in Fig. 6 and Table 4 it follows thattheheat-treatedAg:HA thinfilms depositedonTimodifiedby TiO2nanotubes substrates have a radical antifungal action against thetwo strains, causing the reduction of colonies by 99.73% in case of

A. nigeranda completeextermination incase ofC.albicanscolonies.At the same time, the fungi colonies incubated on control surfaces

where unperturbed and survived integrally.

Identical microbiological tests carried outwith Ag:HA coatingsdirectly depositedonTi substrates (results not shown)evidencedalowerantifungal activity (byseveral tens ofpercents). These results

arein good agreementwithdatapublishedbyLietal.[26] fordentalimplantologic materials (Ti and pure HA among others).

Table 4

Percentageof reductionrate forCandida albicans andAspergillus nigercolonies incu-

batedon theAg:HA/nTiO2/Ti heat-treatedfilms comparedto control.

Sample Candida albicans

(% reduction)

Aspergillus niger

(% reduction)

Control 0 0Ag:HA/nTiO2/Ti 100 99.73

4. Conclusions

We have applied PLD to assemble HA and Ag:HA thin films onthesurfaceof pureTi andTimodifiedby TiO2nanotubes substrates.

A quasi-stoichiometric target-to-substrate transfer was ascer-tained by EDS, whilst the restoration of the crystalline statusafter post-deposition heat-treatment performed at 500 C inwatervapors for 6h was confirmed by the FTIR and XRD analyses. Thus,

benefic effectson thelong termstability of these films inbiologicalfluids shouldbe expected.Wehave shown byAFM that Ag:HA thinfilmsexhibit nanostructured topography which is prone to allow abetter biointegration of the implant.

Dedicated cytotoxicity tests of glass control, Ti substrate,

nTiO2/Ti substrate, and Ag:HA/nTiO2/Ti coating have been con-ducted. The results were at the basis of our choice for conductingthe microbiological antifungal testing on the crystalline Ag:HA

thin films deposited on Ti modified by TiO2 nanotubes substrates.The tests were conducted against two common pathogenic fungalstrains, C. albicansandA. niger. The biological assays demonstratedthe high antifungal efficiency of heat-treated Ag:HA thin films

depositedonTimodified byTiO2nanotubes substrateswhichcom-pletely exterminate C. albicans and radically reduce the A. nigernumber of colonies.

Our conclusion is that the deposition by PLD of Ag:HA thin

films on Ti modified with TiO2 nanotubes substrates followed bya heat treatment at 500 C in water vapors for 6h, allows for thefabrication of efficient shield barriers against adherence and con-tamination by pathogenic fungi.


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Antifungal activity of Ag:hydroxyapatite thin films synthesized bypulsed laser deposition on Ti and...

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