Enhancing the antibacterial performance of orthopaedic implant materials by fibre laser surface ...

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Enhancing the antibacterial performance of orthopaedic implant materials by fibre
laser surface engineering
Chan, Chi-Wai; Carson, Louise; Smith, Graham C.; Morelli, Alessio; Lee, Seunghwan
Published in:
Applied Surface Science
Link to article, DOI:
10.1016/j.apsusc.2017.01.233
Publication date:
2017
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Publisher's PDF, also known as Version of record
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Citation (APA):
Chan, C-W., Carson, L., Smith, G. C., Morelli, A., & Lee, S. (2017). Enhancing the antibacterial performance of
orthopaedic implant materials by fibre laser surface engineering. Applied Surface Science, 404, 67-81.
https://doi.org/10.1016/j.apsusc.2017.01.233

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AppliedSurfaceScience404(2017)67–81

ContentslistsavailableatScienceDirect
Applied Surface Science

journal homepage: www.elsevier.com/locate/apsusc
FullLengthArticle

Enhancing the antibacterial performance of orthopaedic implant

materials by fibre laser surface engineering

Chi-WaiChana,∗,LouiseCarsonb,GrahamC.Smithc,AlessioMorellid,SeunghwanLeee

aBioengineeringResearchGroup,SchoolofMechanicalandAerospaceEngineering,Queen’sUniversityBelfast,BT95AH,UK
bSchoolofPharm acy,Que en’sUn iversity B elfast,BT97 BL,U K
cDepart me ntofNatur alScienc es,Univers ityofC heste r,Th orntonSciencePark,Chester,CH24NU,UK
dCentreforN an ostructu redMedia (CNM),Sc ho olofMa thematics andPhy sics, Queen’sU nive rsity Belfast,BT71NN,UK
eDepart men tofMechanical Engine ering,T echnica lU niversityofD enm ark,DK -2800,K gs.Lyngby, Denma rk

a r t i c l e i n f o a b s t r a c t

Articlehistory: Implantfailurecausedbybacterialinfectionisextremelydifficulttotreatandusuallyrequirestheremoval
Receiv ed23November2016 ofthein fectedc ompon en ts.Despit ethesev er econseque nceofb act erial infe ction,re searchin tob acterial
AReccceepivteedd i2n3 r Jeavnisueadry fo2r0m 1 712 January 2017 in fect ion of ort hopaedic imp lants is stil l at an early stage co mp ared to t he effort o n enhan cing osseoin-
tegration,wearandcorrosionresistanceofimplantmaterials.Inthisstudy,theeffectsoflasersurface
Availableonline25January2017
treatmentonenhancingtheantibacterialpropertiesofcommerciallypure(CP)Ti(Grade2),Ti6Al4V

(Grade5)andCoCrMoalloyimplantmaterialswerestudiedandcomparedforthefirsttime.Lasersur-
Keywords:
facetreatmentwasperformedbyacontinuouswave(CW)fibrelaserwithanear-infraredwavelengthof
Fibrelaser
1064nminanitrogen-containingenvironment.Staphylococcusaureus,commonlyimplicatedininfection
Lasersurfaceengineering
OAnrtthibo apcateedriiacl i pmrpolpaenrttsies atrsesaotceidatseudr fwacitehs .oTrhtheospuarfeadciec riomupglhannetss,s wanads tuospeodg troa pinhvyeosftitghaetela tsheer- atrnetaibteadctmeraiatel rpiarolspweretrieesa onfa ltyhsee dlabseyra-
Staphylococcu saureus 2Droug hnesstes ting andby AFM.Thes urfa cemorpholo gi esb eforeandafte r24hofb acteri alcellcul tur e

werecapturedbySEM,andbacterialviabilitywasdeterminedusinglive/deadstaining.Surfacechemistry

wasanalysedbyXPSandsurfacewettabilitywasmeasuredusingthesessiledropmethod.Thefindings

ofthisstudyindicatedthatthelaser-treatedCPTiandTi6Al4Vsurfacesexhibitedanoticeablereduction

inbacterialadhesionandpossessedabactericidaleffect.Suchpropertieswereattributabletothecom-

binedeffectsofreducedhydrophobicity,thickerandstableoxidefilmsandpresenceoflaser-induced

nano-features.Nosimilarantibacterialeffectwasobservedinthelaser-treatedCoCrMo.
© 2017Elsevie rB.V.Allrightsreserved.

1. Introduction timeconsuming,andmoredifficulttodiagnosethanasepticloos-

eningandusuallyrequirestheremovaloftheinfectedcomponents

Asaconsequenceofthesoaringnumberoftraumacases,e.g. [3].Despitethesevereconsequenceassociatedwithimplantfail-

fromroadaccidentsandsportsinjuries,andwithanincreasingly ure,researchintobacterialinfectionoforthopaedicimplantsisstill

elderlypopulation,thereisastrongglobaldemandfororthopaedic at an early stage compared to the research effort on enhancing

prostheses. A recent market research report indicated that the osseointegration,andonwearandcorrosionresistance.

global orthopaedic implants market was valued at USD 4.3 bil- Bacterial infection is initiated through bacterial adherence to

lion in 2015 [1]. Although significant advancements have been theimplantsurfaces,followedbybacterialcolonizationandbiofilm

made to improve the osseointegration and mechanical proper- formation.Abiofilmisacommunityofmicroorganismsprotected

tiesoforthopaedicimplantsinthepasttwodecades,orthopaedic byaself-producedextracellularpolymericsubstance(EPS)matrix.

implants are still challenged by failures due to various reasons Ithasbeenestimatedthat99%ofbacteriacanexistwithinabiofilm

including aseptic loosening and bacterial infections which con- state[4].Surfacesofdifferentcomponentsoforthopaedicimplants

tributeto30%and16%respectivelyoftotaljointrevisioninthehip suchasthefemoralstem,headandacetabularcuparedesignedfor

andknee[2].Implantfailurecausedbybacterialinfectioniscostlier, differentpurposes.Forexample,thestemandacetabularcup(back

cup),usuallymadeofatitaniumalloy(Ti6Al4V),aredesignedwith

aroughtexturetoencourageosseointegration,whilethefemoral

∗ head,usuallymadeofacobalt-chromiumalloy(CoCrMo),issmooth
Correspondingauthor.
withtheaimofreducingthefrictionbetweenintercalatingcompo-
E-mailaddresses:[email protected],[email protected](C.-W.Chan).

http://dx.doi.org/10.1016/j.apsusc.2017.01.233
0169-4332/©2017ElsevierB.V.Allrightsreserved.
68 C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81

nents.Nevertheless,bacteriacanadheretobothroughandsmooth thereductionofcontactareaintheinterfacebetweenindividual

surfaces, and to different types of materials. Bacterial adherence bacteriumandthemetalsubstrate.

andsubsequentbiofilmdevelopmentcanhampertheperformance However,eachofthestudiesaboveconcernedonlyonepartic-

of the implants, for example by interfering with the process of ulartypeofmaterials(i.e.therewasnodirectcomparisonacross

osseointegration[5].Furthermore,biofilmsareextremelydifficult differentmaterials),andthemajorityofthemusedlaserradiation

toremovewithconventionalantimicrobialtherapies(e.g.antibi- intheultravioletwavelengthrange(i.e.lessthan400nm).Stud-

otics) and act as a reservoir of bacteria that can lead to chronic iesusingnear-infraredlaser(i.e.700–1800nm)forenhancement

and systemic infection [6]. Therefore, strategies to minimise the of antibacterial properties of implant materials are very limited.

chancesofinitialbacterialadherencetotheimplantsurfacesare Intheworkreportedhere,lasersurfacetreatmentwasperformed

crucialtopreventbacterialinfection. onthreecommonly-usedorthopaedicmetallicmaterials,namely

Bacterialadherencetoasurfaceisdependentonseveralinter- CPTi(grade2),Ti6Al4V(grade5)andCoCrMousingafibrelaser

relatedsurfacepropertiesofmaterials,suchassurfaceroughness, withanear-infraredwavelengthof1064nminanitrogenshield-

topography,chemistryandwettability.Bacteriaprefertoadhereto ingenvironment.ItisknownthatTiNformsonthesurfacewhen

aroughsurfacethanasmoothsurface[7,8],andtohydrophobic Ti-materialsreactwithhighpowernear-infraredlaserinthenitro-

rather than hydrophilic surfaces [9,10], while nano-scale surface genenvironment.TiNisahighlywear-resistantandbiocompatible

features are more effective in reducing bacterial adhesion than material [15]. S. aureus, the most common organism responsible

micro- and macro-scale features [11,12]. Material chemistry can for orthopaedic surgical site infections [20], was selected as the

alsoinfluencebacterialcolonizationofamaterialsurface,forexam- targetbacteriainthestudy.Theobjectivesofthisstudyare(1)to

plecertainmetalions,e.g.silver(Ag),carbon(C),zinc(Zn),copper comparetheantibacterialeffectofdifferentorthopaedicmaterials

(Cu),andsomemetaloxides/nitrides,e.g.,titaniumdioxide(TiO ), beforeandafterlaser-treatmentwithnear-infraredradiation,and
2

zinc oxide (ZnO), tantalum nitride (TaN), titanium nitride (TiN), (2)toexplainthedifferenceinantibacterialperformancebetween

and zirconium nitride (ZrN) [9] all exhibit intrinsic antibacterial treatedanduntreatedsurfacesintermsofthesurfaceroughness,

properties. topography,chemistryandwettability.

Strategiestoreducebacterialadherencecangenerallybeclas-

sified into coating and non-coating methods. The basic concept
2. Experimentaldetails
of coating methods is to coat the entire implant with the afore-

mentionedantibacterialmaterials.However,thedrawbackofusing
2.1. Materials
antibacterialmaterialsisthepossibilityofcytotoxicitytothehost

cellsandtissues.Forexample,copperisknowntodisplaycytotox-
Threedifferentmedicalgrademetallicmaterialswereusedfor
icity towards mesenchymal stem cells [2]. Non-coating methods
the laser treatment experiments, namely commercially pure Ti
directly modify the surface properties of implants to achieve
(99.2% pure, Grade 2) and Ti6Al4V (Grade 5), and CoCrMo alloy.
antibacterialcharacteristics.Thesemethodsincludereducingsur-
TheyweresourcedfromZappPrecisionMetalsGmbH(Schwerte,
facehydrophobicity[10],creatingsurfacenano-features[12]and
Germany).TheGrade2andGrade5titaniummaterialsarelabeled
modifyingsurfacechemistry[10,13,14].
asTiG2andTiG5hereafter.Thesampleswereintheformofdiscs
Lasersurfacetreatmentisemergingasapromisingnon-coating
30mmindiameterand5mminthickness.Beforelasertreatment,
method to negate bacterial adherence. The advantages of laser
the sample surfaces were ground sequentially with a series of
technology include high speed, cleanliness, high precision and
SiC papers from 120 to 400 grits following standard metallogra-
repeatability, as well as flexibility to modify surfaces in selec-
phyprocedurestoremovepre-existingoxidesandensuresurface
tiveareas[15].Further,lasertechnologycanbeusedalongwith
homogeneity. The samples were then ultrasonically cleaned in
three-dimensional (3D) printing technology. The laser-based 3D
ethanolbathfor10min,rinsedindistilledwaterforanother10min,
printing technique of selective laser sintering (SLS) has recently
andfinallydriedthoroughlyinacoldairstream.
beenappliedtofabricatebonescaffoldswithantibacterialproper-

ties[16,17].
2.2. Lasertreatmentexperimentsinnitrogenenvironment
Recent successful examples of using laser surface treatment

techniquestofabricateantibacterialsurfacesformetallicimplant
Thelasertreatmentprocesswasperformedusinganautomated
materialsarereviewedasfollows:Gallardo-Morenoetal.[10]used
continuouswave(CW)200Wfiberlasersystem(MLS-4030).The
UV irradiation at a wavelength of 258nm to treat Ti6Al4V alloy.
lasersystemwasintegratedbyMicroLasersystemsBV(Driel,The
TheirresultsindicatethatthephysicochemicalchangesontheUV
Netherlands)andthefibrelaserwasmanufacturedbySPILasersUK
treatedsurfacecausedareductionintheadhesionrateofStaphy-
Ltd(Southampton,UK).Thewavelengthofthelaserwas1064nm.
lococcusaureusandStaphylococcusepidermidiscells.Kawanoetal.
Thesampleswereirradiatedwiththelaserbeamusingpre-selected
[18]usedaUVlaserwithawavelengthof365nmtotreatcom-
processingparametersof:laserpowerof40W,scanningspeedof
mercially pure (CP) Ti. Their study suggests that exposure of CP Ti 25mm/s(m eanderedsc an with lateralm o vem en tof100(cid:2) mint he
toaUVlasercandecreasethenumberofattachedPorphyromonas
xdirection),stand-offdistanceof1.5mm(laserspotsizewasmea-
gingivalis bacterial cells, this bacterium being an important cause su redas100 (cid:2)m)and shieldin gw ith hig hpuri tyN at5 bar [21].
ofdentalimplantinfections.Theyascribedtheantibacterialeffect 2
TheN gaswasdeliveredcoaxiallywiththelaserbeamviaastan-
tothedecreaseofwatercontactangleandincreaseoftheAnatase 2
dardlasernozzlewithoutletdiameterof2mm.Thelaser-irradiated
phase in the surface layer on treated Ti surface. Gillett et al. [19] area onth edisks amp leswas 18mm× 1 8 mma nd fullycoveredby
employedanexcimerlaserwithawavelengthof248nmtosur-
lasertrackswithoverlappingratioofabout50%intrackwidth.
facepatternpolyethyleneterephthalate(PET).Theyreportedthat

thesurfacetreatmentcreatedmicro-scalepitsinsurfaceandsig-

nificantlyinfluencedthedistributionandmorphologyofattached 2.3. Surfacemorphology,roughnessandtopographyanalysis

Escherichiacolicells.Cunhaetal.[5]creatednano-featuresonCPTi

surfaceusingafemtosecondlaserwithawavelengthof1030nm. Thesurfacemorphologyoftheuntreatedandlaser-treatedsam-

Theyfoundthatthenano-topographyofthelaser-inducedfeatures ples was captured using a scanning electron microscope, SEM

reduced adhesion of S. aureus cells, and attributed the effect to (Model 6500F, JEOL, Japan). The surface roughness and topogra-

phy of the untreated and laser-treated samples were assessed
C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81 69

using a portable roughness gauge (Rugosurf 10G, Tesa Technol- withshakingat100rpm.Afterincubation,sterileMHBwasusedto

ogy)andacommercialatomicforcemicroscope(AFM)intapping adjusttheovernightculturetoanopticaldensityof0.3at550nm

mode(D5000,VeecoDigitalInstruments).Thesurfaceroughness anddiluted1in50withfreshsterileMHB.Thisprovidedabac-
tester was use d to m easure the 2D large ste p surfa ce profiles teria l inocu lu m of a ppro ximat ely 1× 106 C olony Forming U nits

(in macro-scale) whilst the AFM served to characterize the 3D (CFU)/ml.1mlofculturewasappliedtoeachsampleataninocu-
mic ro/nano-scale featur es in loca l areas of t he surface. T he s can lumnotex c eed ing 2.4×1 06CF U/ml,as ve rified byviab lec ou nt.The
lengthofthesurfa cerough ne sstes terwa s1 5m mwhils tthe scan sam ples wereincu bat ed for24hat3 7◦ Conag yro tatory incuba tor
size of th e A FM was 2(cid:2)m×2(cid:2) m. B asic rou ghne ss par ame ters, with sha king at 100rpm . T hre e sa mple s o f each type o f materi-

namelyRa(arithmeticmeanroughness)andRz(maximumheight als,forbothuntreatedandlaser-treated,weretestedtoensurethe

ofprofile)weremeasuredusingthesurfaceroughnesstester.At consistencyoftheresults.

least12measurementsweretakenatdifferentlocationsforeach

sampleinthedirectionperpendiculartothelasertrackorientation. 2.7. Bacterialviabilityanalysis

Theadditionalsurfaceroughnessparameters,namelyRsk(skew-

nessofprofile)andRku(kurtosisofprofile)weremeasuredbyAFM. After24hofincubation,thesampleswerewashedthreetimes

Atleasttwomeasurementsweretakenforeachsample.Theloca- withsterilePBStoremoveanynon-adherentbacteria.Theadher-
tio nsof mea surementswere rand omlys elec tedf romtheu ntr eated entb acteria wer es tainedw ith fluorescentLiv e/Dead® BacL ightTM
surfa ces whilstthemea surem entswere takenat there gio nnearthe solu tion(Mo lecula rProbe s)for 30minat37 ◦Cinthedark.Theflu-

centrelineoflasertracksinthelaser-treatedsurfaces.Topographic orescent viability kit contains two components: SYTO 9 dye and

analysiswasperformedviatheWSxMsoftware[22]. propidiumiodide.TheSYTO9labelsallbacteria,whereaspropid-

iumiodideentersonlybacteriawithdamagedmembranes.Green

2.4. Surfacewettabilityanalysis fluorescenceindicatesviablebacteriawithintactcellmembranes

whilst red fluorescence indicates dead bacteria with damaged

Thesessiledropmethodwasusedtomeasurethecontactangle membranes. The labelled bacteria were observed using a fluo-

ofaliquiddropontheuntreatedandlaser-treatedsamplesusinga rescence microscope (GXM-L3201 LED, GX Optical). At least ten

video-basedcontactangleanalyzer(FTA200,FirstTenAngstroms). random fields of view (FOV) were captured per sample. The

TheimagecaptureandanalysiswereperformedusingtheFTA32 surface areas covered by the adherent bacteria were calculated

video software. Deionized water was used as the testing liquid, usingtheImageJsoftware(developedattheNationalInstitutesof
andth evolume ofeachse ssiledr opw asco ntr olle dat5(cid:2) lusing Health , B ethesda , Marylan d, U.S.)(htt ps: //im agej.nih. gov/ij/). T he

amicrolitresyringe.Dropletimageswerecapturedinthedirection areas corresponding to the viable bacteria (coloured green) and

perpendiculartothelasertrackorientationatfixedtimeintervals, thedeadbacteria(colouredred)wereindividuallycalculated.The

countingsincethestartofdropletdepositiontothecessationof totalbiofilmareawasthesumofthegreenandredareasandthe

dropletspreadingoratleast60s.Atleasteightmeasurementswere dead/livecellratiowastheratiobetweenthegreenandredareas.

takenatdifferentlocationsforeachsampleatroomtemperature. Theresultswereexpressedasthemeansofmeasurementsfrom

thetenimages.

2.5. Surfacechemistryanalysis

2.8. Bacterialmorphologyanalysis

X-rayphotoelectronspectroscopy(XPS)spectrawereacquired

usingabespokeultra-highvacuumsystemfittedwithamonochro- After removing samples from the bacterial culture, the sam-
mated Al K(cid:3) X- ray source (Specs GmbH Focus 500, B erlin) with pleswer einitiallyr insedwi th0.9 %sal inefor1m intore mov eany

aphotonenergyof1486.6eV,150mmmeanradiushemispheri- non-adherentbacteria.Thisprocesswasrepeatedthreetimes.The

cal analyser with 9-channeltron detection (Specs GmbH Phoibos adherent bacteria were then fixed using 2.5% glutaraldehyde in

150,Berlin),andachargeneutralisingelectrongun(SpecsGmbH 0.1Mcacodylicacid(pH7.2).Thesampleswerekeptinthissolution
FG20 ,Berlin) .The a nalysis areawasap proximat ely2 mmin diam- for 24 hat4◦C. Afte rthe fixa tion ,thesam ples were de hyd ratedin

eter. Survey scans were acquired over the binding energy range a graded series of ethanol: 50%, 70%, 90% and 100% with 30min

between0and1100eVusingapassenergyof50eV,andthehigh- each at room temperature. The dehydrated samples were then

resolutionscansovertheTi2p(forTiG2andTiG5),Al2p(forTiG5), transferredtoa24-wellplatecontainingadyingagentofhexam-

Co2pandCr2p(forCoCrMo),andN1s(foralltypesofsample) ethyldisilazane(HMDS)andlefttodryfor24hinafumecupboard.

linesweremadeusingapassenergyof15eV.Datawerequantified Thesamplesweresputter-coatedwithAuforbacteriamorphology

usingScofieldcross-sectionscorrectedfortheenergydependen- observationbySEM(Model6500F,JEOL,Japan).Thecleansamples

ciesoftheeffectiveelectronattenuationlengthsandtheanalyser wereimagedbySEMasacontrol.

transmission. Data processing and curve fitting were carried out

usingtheCasaXPSsoftwarev2.3.16(CasaXPS,Teignmouth,UK). 2.9. Statisticalanalysis

2.6. Bacterialcellculture Thesignificanceoftheobserveddifferencesbetweenthemeans

of different samples were analysed and compared by one-way

Boththelaser-treatedanduntreatedcontrolsamplesofTiG2, ANOVA and Tukey’s test using SPSS software (version 19, SPSS,

TiG5 and CoCrMo alloys were used for bacterial adherence and Inc.).Theprobabilitybelowp<0.05wasconsideredasstatistically

biofilmformationassays.Thesampleswerecutintheformofcir- significant.

cular discs with 8mm diameter by electric discharge machining

(EDM). Samples were cleaned with pure ethanol (Sigma Aldrich, 3. Results

UK)inanultrasonicbathfor15minpriortobacterialcellculture.

Thedry,cleansampleswerethenplacedintoa24-welltissuecul- 3.1. SurfacemorphologybySEM

tureplate.Theywerethensterilizedwith70%ethanolfor10min

and washed three times with sterile phosphate buffered saline TheSEMmicrographsfortheuntreatedandlaser-treatedsur-

(PBS).S.aureus(ATCC6538)wasculturedinMüllerHintonBroth facesareshowninFig.1(a–i).Atypicalsurfacemorphologyafter
(MHB; O xoid)o vernig ht(18 h)at 37◦Con a gyrotat oryincu bator mech anic algrind in gca nbeob se rvedfro mtheu ntreatedsur faces
70 C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81

Fig.1. (a–i).SEMmicrographsforthe(a–c)untreatedand(d–i)laser-treatedsamples:TiG2(a,d&g),TiG5(b,e&h)andCoCrMo(c,fandi).Themicrographsfrom(a)to(f)

areatlowermagnificationof1000,whilstthemicrographsfrom(g)to(i)arethemagnifiedviewsof(d)to(f)at3000.

of TiG2, TiG5 and CoCrMo, showing the presence of randomly- 3.2. Surfaceroughnessby2Droughnesstester

oriented scratch marks (Fig. 1a–c). As observed in Fig. 1d–f, all

laser-treatedsurfaces,namelyTiG2,TiG5andCoCrMo,showcircu- TheRaandRzvaluesfortheuntreatedandlaser-treatedsamples

larrosette-likemarkings.Suchrosette-likemarkingswerecreated extractedfromthe2DroughnessprofilesaregiveninTable1.TheRa
asa consequen ceofthem oving laserbeam (operated atCW mode) valuesfor theu ntr eat edsamples areint her ange of 0.04– 0.3 6(cid:2) m,
“st o pping”ateac h loca tionon them etals urfacefor a very short whilet he Rzv aluesare between 0.33 a nd2 .58(cid:2) m. TheRaan dRz

periodoftimeduringthelasertreatmentprocess,allowingthelaser values of the untreated samples follow the same order with the

beamtomeltandinteractwiththemetal[21].Usingtheempirical untreatedTiG2beingthehighest,followedbytheuntreatedTiG5

equationderivedbySuderandWilliams[23],theinteractiontime andthelowestistheuntreatedCoCrMo.BoththeRaandRzval-

(i.e.laserspotdiameterdividedbyscanningspeed)betweenthe ues increase significantly after laser treatment, with the Ra and
lase rbeam and themeta lsurface wa scalculat edas4 ms.Them ag- Rzv alueslyin gbetween1 .82an d3.56 (cid:2)mandbe twee n10 .85 and
nified view sin Fig. 1gan dhsho wth attherose tte -l ike mar kings 18 .70(cid:2)m ,resp ectively. Howe ver, the orde ris different from that

inlaser-treatedTiG2andTiG5surfacesconsistofsecondarymicro- whichisobservedinthegroupoftheuntreatedsamples,withthe

/nano-sizedfeaturessuchasripplesandradiallamellae(seearrows laser-treatedTiG5beingthehighest,followedbythelaser-treated

inFig.1gandh).Theripplefeatures(seearrowinFig.1i)canstill TiG2andthelowestisthelaser-treatedCoCrMo.Itcanbeobserved

befoundintherosette-likemarkingsoflaser-treatedCoCrMobut thattheTiG2andTiG5samplesintheuntreatedandlaser-treated

radiallamellaeareabsentfromthesurface. groupshavehigherRaandRzvaluesthantheCoCrMosample.
C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81 71

Table1

Summaryofthesurfaceroughnessparametersandwatercontactanglesfortheuntreatedandlaser-treatedsamples:TiG2,TiG5andCoCrMo.

Samples SurfaceRoughnessParameters SurfaceWettability

Ra((cid:2)m) Rz((cid:2)m) Rsk Rku WaterContactAngle(◦)

Measuredby2DSurfaceRoughnessTester MeasuredbyAFM MeasuredbySessileDropMethod

UntreatedTiG2 0.37 2.59 −0.34 2.06 74.3

UntreatedTiG5 0.13 1.14 0.22 2.29 72.6

UntreatedCoCrMo 0.04 0.33 80.6

Laser-treatedTiG2 2.60 18.70 0.07 3.04 31.9

Laser-treatedTiG5 3.57 23.23 0.32 3.11 45.7

Laser-treatedCoCrMo 1.83 10.86 83.0

Fig.2. (a–d).AFMtopography3Dimagesforthe(a-b)untreatedand(c-d)laser-treatedTiG2andTiG5.Scanarea2×2(cid:2)m2;colorcodingofallimagesasbyheightscalein

figure.
3.3. SurfacetopographybyAFM untreatedTiG2andTiG5surfaceswereusedascontrol.Thesec-

ondarysurfacefeatureswerequantifiedbytwoadditionalsurface

The secondary surface features in the laser-treated TiG2 and roughnessparameters,namelyRskandRku.TheRskandRkuval-

TiG5(refertoFig.1gandh)werefurtheranalysedusingAFM.The uesfortheuntreatedandlaser-treatedTiG2andTiG5aregivenin
72 C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81

laser-treatedCoCrMoissimilartoeachother.Theorderofsurface

hydrophobicityforthelaser-treatedsamplesis:

Laser-treatedCoCrMo>Laser-treatedTiG5>Laser-treatedTiG2.

3.5. XPSsurveyscans

Thesurfacecompositionresultsinatom%,excludingHandHe,

andnormalisedto100%ofelementsdetected,areshowninTable2.

Anumberofobservationscanbemadefromthesurveyscanresults.

ComparingresultsforTiG2andTiG5,thelevelsofTiarereduced

bylasertreatment,slightlyinthecaseofTiG2andbymorethan

half in the case of TiG5. In both materials, surface oxygen levels

arereducedbutnitrogenlevelsareincreasedafterlasertreatment.

Thissuggeststhatthenativesurfaceoxidewasreplacedbyasec-

ondsurfacefilm(e.g.TiN)duringlasertreatment.Carbonispresent

ontheuntreatedandlaser-treatedsamplesintheformofadven-

titioushydrocarbon,i.e.residualcontamination.Additionally,TiG5

showsAlandVontheuntreatedsurface.However,thesurfaceratio

ofTi,AlandVisfarfromthatofthebulkcomposition,indicating

surfaceenrichmentinAl.Afterlasertreatment,thissurfaceenrich-

mentisevenmorepronounced,withconsiderablymoreAlthan

Tiatthesurface.ItisparticularnoteworthythatnoVisdetected
Fig. 3. Mean contact angles of water (n = 8) with error bars (95% confident inter- fro m lase r-treate dT iG 5.AsforC oCrMo,both untr eat ed a ndlaser-
val)forthetwogroupsofsamples:untreatedandlaser-treatedsamples.Withinthe
treatedsurfacesshowhighlevelsofcarbon.ThelevelsofCoandCr
groupoftheuntreatedsamples,nosignificantdifferencesareobserved.Withinthe
group of the laser-treat edsample s,t helaser-tre atedCoCrM oh asthehigh estcon tact areslightlyincreasedafterlaser-treatment.However,theCoandCr
angle, fo llow edbythelas er-treated TiG 5andthelas er-treate dT iG2 hasthe lowest. leve lsarera therlowi nboth surfaces,probab lyasares ulto fa tten u-
Result sarestat isti call ysignificant atp< 0.05 .A statisticallys ignifi can tre duction ationo fth esign alby th ehyd rocarbon overlaye r. Th eoxy ge nlevels
inthec onta ctangleiso bservedin the l as er-tr ea tedTiG2and TiG5whe ncompar-
areincreasedwithlasertreatment,asarethelevelsofCrandCo.
ingwiththeiruntreatedcounterparts.Nostatisticallysignificantdifferenceexists
sbiegtn wifieceann tthaet up n<tr0e.0at5e.d a nd laser-treate d C rCrMo.*indi cates the m ean differe nce is Ntreevaetmrthenelte.ss, no increase is found in the nitrogen levels after laser

3.5.1. NarrowscanofN1sspectrum(forTiG2,TiG5andCoCrMo)
Table1,andtheir3DsurfaceprofilesareprovidedinFig.2(a–d).
HighresolutionscansovertheN1slineontheuntreatedand
Rku is a measure of the sharpness of the profile. Spiky surfaces
laser-treated surfaces of TiG2, TiG5 and CoCrMo are shown in
haveahighkurtosisvalue(Rku>3)whereasbumpysurfaceshave
Fig.4(a–f).TwopeaksareseenfromTiG2(Fig.4aandb)andTiG5
alowkurtosisvalue(Rku<3).Rskdescribestheasymmetryofa
(Fig.4candd).Thecomponentat396eVisduetonitrogeninthe
surface. A negative skewness value indicates a predominance of
formofnitride;thatat400eVisduetonitrogeninanelectronically
troughswhereasapositiveskewnessvalueindicatesanabundance
neutralformtypicalofanorganicspecies(e.g.amine-typebonding)
ofpeaks.
andisattributedtothegenerallow-levelenvironmentalcontami-
TheresultsinTable1indicatethatuntreatedTiG2andTiG5have
nationexpectedonair-exposedsurfaces.Comparinguntreatedand
alowRkuvalue(<3)butTiG2hasanegativeRskwhilstTiG5hasa
laser-treatedTiG2,theresultsindicateasubstantialincreaseinthe
positiveRsk.Incomparison,boththelaser-treatedTiG2andTiG5
nitridecomponentonlaser-treatedTiG2(Fig.4b)andthisincrease
haveapositiveRskandahighRku(>3).Theresultsofskewness
in the nitride component accounts for the increase in the total
andkurtosisanalysispointtothefactthatbothuntreatedTiG2and
nitrogenlevelfrom3.9%to7.7%showninTable2.Likewise,asub-
TiG5tendtohaveabumpysurface(Fig.2aandb)butuntreated
stantialincreaseintherelativeproportionofthenitridecomponent
TiG2hasmoretroughsthanpeaksthoughtheoppositeisfoundin
isobservedonlaser-treatedTiG5(Fig.4d)buttheincreaseinthe
untreatedTiG5.Laser-treatedTiG2andTiG5tendtohaveaspiky
totalamountofnitrogenfrom2.7%to3.6%isnotasgreatasseenfor
surface(Fig.2candd)withmorepeaksthantroughsinthesurface,
laser-treatedTiG2(Table2).RegardingCoCrMo(Fig.4eandf),only
i.e.duetothepresenceofsecondarysurfacefeatures.Asseenin
onepeakat400eVisseenontheuntreatedsample(Fig.4e).On
Fig.2candd,thesecondarysurfacefeaturesonlaser-treatedTiG5
laser-treatedCoCrMo(Fig.4f),theN1sspectrumshowsthepres-
arenoticeablysmallerandspikierthanthoseonlaser-treatedTiG2.
enceofafurthercomponentat397eV,anenergytypicalofametal

nitrideform.However,theproportionofthenitridecomponentis

3.4. Surfacewettabilitybysessiledropmethod verysmall,andthusthereisnotenoughevidencetosupportthe

existenceofasignificantnitridefilmonlaser-treatedCoCrMo.

The water contact angles on the untreated and laser-treated

samples are given in Table 1 and plotted in Fig. 3. It has been 3.5.2. NarrowscanofTi2pspectrum(forTiG2andTiG5)

reportedthatmaterialsurfacescanbeconsideredhydrophobicif TheTi2pspectrafromtheuntreatedandlaser-treatedsurfaces
thewate rcon tactangle islarger tha n5 0◦[24].From theresults in of TiG2 a nd TiG5 a re sh ow n in Fig. 5(a– d). Each che mically-
Tab le1,al luntreat edsam p leshav eah ighconta ctang leof over70 ◦, sh ifted Ti 2p peak is split in to 2p and 2p d oublets. The
3/2 1/2
indicatingthatuntreatedTiG2,TiG5andCoCrMoarehydrophobic. various chemically-shifted components of the Ti 2p peak are
Ontheoth erh and,thew aterc ontac tan glesonla ser -treatedTiG2 found a s follows: Ti0 or metallic Ti= 454 eV, Ti O=34/255eV, TiN

andTiG5arefoundtoberemarkablysmaller,withstatisticalsignif- or Ti O =455.8eV and TiO =458.8eV. The spectrum from the
2 3 2

icance(p<0.05).Thisindicatesthatthesurfacehydrophobicityof untreatedTiG2surface(Fig.5a)isdominatedbyastrongTiO com-
2

TiG2andTiG5isgreatlyreducedafterlasertreatment.Furthermore, ponent, as expected for an air-exposed Ti surface. Metallic Ti is

thedecreaseinthecontactangleismoreprofoundforlaser-treated visible,indicatingthatthesurfaceoxidefilmisprobablyverythin
TiG 2thanlas er -tre atedTiG 5.Th e conta ctangleo nu ntreatedand (∼fewn m). Low l evels of interme diate state s including TiO and
C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81 73

Table2

Summaryofatom%compositionsbyXPSontheuntreatedandlasertreatedsamples:TiG2,TiG5andCoCrMo.

TiG2 TiG5 CoCrMo

Elements&lines Untreated Laser-treated Untreated Laser-treated Untreated Laser-treated

Ti2p 9.0 8.1 3.8 1.5 –– ––

Al2p –– –– 5.4 12.5 –– ––

V2p3/2 –– –– 0.2 –– –– ––

Co2p3/2 –– –– –– –– 1.8 2.7

Cr2p3/2 –– –– –– –– 1.6 2.6

O1s 32.8 28.0 23.3 23.0 13.1 19.6

N1s 3.9 7.7 2.7 3.6 2.9 1.7

C1s 50.1 53.7 62.1 55.6 80.7 72.0

Others:Ca2p,Cl2p,Fe2p,P2s,S2p,Si2p,Zn2p3/2 4.3 2.5 2.5 3.8 0.0 1.5

Fig.4. (a-f).XPSN1sspectrafromtheuntreatedandlaser-treatedsamples:. (a-b)TiG2–N1s,(c-d)TiG5–N1sand(e-f)CoCrMo–N1s
74 C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81

Fig.5. (a-f).XPSTi2pandAl2pspectrafromtheuntreatedandlaser-treatedsamples:. (a-b)TiG2–Ti2p,(c-d)TiG5–Ti2pand(e-f)TiG5–Al2p

TiN/Ti O arealsovisible.Afterlasertreatment,thespectrumof theTiO componentafterlasertreatmentisconsiderablyweaker
2 3 2

TiG2(Fig.5b)showsnometallicTi.AsaproportionofthetotalTi thanthatinthelaser-treatedTiG2.

signal,theTiO componentislowerandtheTiNandTiOcompo-
2

nentsarestrongerthanwerefoundonuntreatedTiG2.OnTiG5,the
3.5.3. NarrowscanofAl2pspectrum(forTiG5only)
spectrumoftheuntreatedsurface(Fig.5c)looksverysimilartothat
The Al 2p spectra from untreated and laser-treated TiG5 sur-
fromtheequivalentareaofuntreatedTiG2.TheTi2pregionisdom-
ainnadteT di3 b+y a rsetraolnsog dTeiOt e2ccteod m.pT honeeTni3t+s,c aon mdp loonw e nletv heal ss ob fe Teni matet traib l,u Ttie2d+ fsaucrefas caerse, sthhoew snp ienc tFriag . 5ar(ee– fd)o. Omnin baottehd ubnytr emataejdor a ncdo mlapseorn-etrnetast eadt
approximately74.2–74.4eV,correspondingtoAlinitshighestoxi-
tmoi xTeiNd,o axsy -nnititrrididee icso smeepno ninen tthcea nNn o1t sb sepreuclterda,o buuttf rToim2Ot3heosre sdoamtae. dation state, A l2O3 or p ossi bly Al(OH)3. O n the u nt reated T iG5
(Fig.5e),thereissomeevidenceforaweakcomponentatapprox-
Onthelaser-treatedTiG5surface(Fig.5d),thespectrumshowsno
imately 71.6eV corresponding to Al in a metallic alloy form. On
metalliccomponent,asisthecaseforlaser-treatedTiG2.However,
the laser-treated TiG5 (Fig. 5f), the effect of the laser treatment

appears to have been to convert a thin oxide film where some
C.-W.Chanetal./AppliedSurfaceScience404(2017)67–81 75

Fig.6. (a-d).XPSCo2pandCr2pspectrafromtheuntreatedandlaser-treatedsamples:. (a-b)CoCrMo–Co2p,and(c-d)CoCrMo–Cr2p

metalliccharactercouldbedetected,intoafullyformedoxide.In greenandredcoloursindicatethebacterialcellswithintact(live)

thiscase,asubstantialincreaseintheamountofAlatthesurface anddamagedmembrane(dead),respectively.Asseeninthefigures,

isalsodetected,possiblyindicatingmigrationorsegregationtothe ahighamountofgreenfluorescenceisfoundfromthesurfacesofall

surface and subsequent oxidation as a consequence of the laser untreatedsamples,namelyTiG2(Fig.7a),TiG5(Fig.7c)andCoCrMo

treatment. (Fig.7e),indicatingahighnumberofviablebacterialcellsadhered

on the surfaces. The viable cells are able to adhere and grow on

theuntreatedsurfacesandaggregatetoformthebiofilms.Incom-
3.5.4. NarrowscanofCo2pandCr2pspectrum(forCoCrMoonly)
parison,thenumberofviablebacteriaonthelaser-treatedTiG2is
TheCo2pandCr2pspectrafromtheuntreatedandlaser-treated
reducedremarkably(Fig.7b),i.e.onlyasmallamountofgreenflu-
CoCrMosurfacesareshowninFig.6(a–d).TheCospectralrange
orescenceisevident.Inadditiontothereducednumberofviable
(Fig. 6a and b) shows a doublet consisting of 2p3/2 components cells,reds ta ining(no n- viableba cte ria) isdomin antont he laser-
in the binding energy range 775–790 eV and 2p1/2components in treate d T iG2. Sim ilar observa tions are se en on the las er-tr eated
the energy range 791–808 eV. The Co 2p3/2 spectral region from TiG5(F ig.7d) .These findingsindic atet hatth el aser -treatedTiG2
theuntreatedsurface(Fig.6a)showsasharpandrelativelyintense
andTiG5surfacesmaybelesshospitableforS.aureusadherenceand
componentat778eVandabroadfeatureat781eV.Thesharpfea-
maypossessabactericidaleffect.Incontrast,green-fluorescence
tureat778eVisduetothemetallicCoandthefeatureat781eVis
from viable bacteria was dominant on the laser-treated CoCrMo
duetooxidisedCointheformofCoO.Onthelasertreatedsurface
surface(Fig.7f).
(Fig.6b),thereisnoevidenceformetallicCo.TheCoappearsfully
oxid ised inthe C o2+ form.Fur the rmore,t heC r2p sp ectra(Fi g.6c

andd)mirrorthebehaviouroftheCo2p.Ontheuntreatedsurface
3.7. Statisticalanalysisofbacterialadhesionandbactericidal
(Fig.6c),thespectrumshowsthemetallicCrandtheoxidisedCrin
effect
theformofCr O .Onthelaser-treatedsurface(Fig.6d),onlythe
2 3

oxidecomponentsareseen,i.e.noevidenceforthemetallicCr.
Thebacterialadhesionisquantifiedbycalculatingthepercentof

totalbiofilmarea.TheresultsareplottedinFig.8.Theresultsshow

3.6. Fluorescenceimages(Live/Deadstaining)ofbiofilms thatthelaser-treatedTiG2andTiG5surfacesexhibitedanoticeable

reductioninbacterialadhesionwhencomparedtotheiruntreated

TheimagesobtainedbyfluorescencemicroscopyinFig.7(a–e) counterparts. The results are statistically significant at p<0.05.

showstheadhesionofS.aureusontheuntreated(Fig.7a,cande) However,therewasnostatisticallysignificantdifferencebetween

andlaser-treated(Fig.7b,d,andf)surfacesafter24hculture.The theuntreatedandlaser-treatedCoCrMoinbacterialadhesion.The