Review of Antibacterial Activity of Titanium-Based Implants' Surfaces Fabricated by Micro-Arc ...

22 Pages·2017·15.16 MB·English

coatings
Review
Review of Antibacterial Activity of Titanium-Based
Implants’ Surfaces Fabricated by Micro-Arc Oxidation
XiaojingHe,XiangyuZhang*,XinWangandLinQin
CollegeofMaterialsScienceandEngineering,TaiyuanUniversityofTechnology,Taiyuan030024,China;
[email protected](X.H.);[email protected](X.W.);[email protected](L.Q.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:YuelianLiu
Received:28November2016;Accepted:14March2017;Published:22March2017
Abstract: Ti and its alloys are the most commonly-used materials for biomedical applications.
However, bacterial infection after implant placement is still one of the significant rising
complications.Therefore,theapplicationoftheantimicrobialagentsintoimplantsurfacestoprevent
implant-associated infection has attracted much attention. Scientific papers have shown that
inorganicantibacterialmetalelements(e.g., Ag, Cu, Zn)canbeintroducedintoimplantsurfaces
with theaddition of metalnanoparticles or metalliccompounds intoan electrolyte via micro-arc
oxidation (MAO) technology. In this review, the effects of the composition and concentration of
electrolyteandprocessparameters(e.g.,voltage,currentdensity,oxidationtime)onthemorphological
characteristics(e.g.,surfacemorphology,bondingstrength),antibacterialabilityandbiocompatibility
of MAO antimicrobial coatings are discussed in detail. Anti-infection and osseointegration can
be simultaneously accomplished with the selection of the proper antibacterial elements and
operating parameters. Besides, MAO assisted by magnetron sputtering (MS) to endow Ti-based
implantmaterialswithsuperiorantibacterialabilityandbiocompatibilityisalsodiscussed.Finally,
thedevelopmenttrendofMAOtechnologyinthefutureisforecasted.
Keywords: micro-arcoxidation;antibacterialability;Ag;Cu;Zn
1. Introduction
Biomedicaltitanium(Ti)anditsalloyshavebeenwidelyusedinbloodvessels,artificialjoints,
dentalimplantationsandbonescrews[1–3]onaccountoftheirexcellentmechanicalproperties,good
corrosion resistance and favorable biocompatibility. However, implant-associated infection (IAI)
remainsoneofthemostdevastatingpostoperativecomplications[4,5]despitestrictsterilizationand
systemic antibiotic prophylaxis prior to surgery. IAI often commences with bacterial adhesion to
theimplantandcolonizationontheimplantsurface, followedbybiofilmformation. Thebiofilms
are extraordinarily resistant to antibiotics and the host immunity defensive system [6–8], leading
to further complications. Once the biofilm is formed, it is often not effectively treated except for
prosthesisremovalandre-implantation[9,10]. Thisdevastatingcomplicationmayresultinchronic
sufferingandextremelyhugemedicalexpenses[11,12]. Therefore,itishighlydesirabletointroduce
antimicrobialagentsintoimplantsurfacestoprovideantibacterialactivitiesandpreventperi-implant
infections [13–17]. In comparison with organic antibiotics, inorganic antibacterial metal elements
(e.g.,silver(Ag)[18–22],copper(Cu)[23–26]andzinc(Zn)[27–29])haveattractedgreatattentiondue
totheirperfectstabilities,superiorbroad-spectrumantibacterialproperties,relativelylowtoxicityto
humancellsandlowriskofproducingresistantstrains[30,31].
Dizajetal.[32]havereviewedtheantimicrobialactivityofmetalandmetaloxidenanoparticles
togetherwiththeirantimicrobialmechanismsandhaveindicatedthattheparticlesizedetermined
theantimicrobialeffectivenessofthemetalnanoparticles. Raietal.[33]haveextensivelyreviewed
Coatings2017,7,45;doi:10.3390/coatings7030045 www.mdpi.com/journal/coatings
Coatings2017,7,45 2of22
theantibacterial,antifungalandantiviralpropertiesofAgions,AgcompoundsandAgnanoparticles,
butthereisnodiscussionabouttheantibacterialpropertiesofCuorZn. Inaddition,thetoxicities
ofmetalandmetaloxidenanoparticlestoapplyasproperalternativesforantibioticsinbiomedical
applications were not discussed in the reviews. In order to obtain antibacterial metallic surfaces,
numeroussurfacemodificationshavebeenperformed. Ferrarisetal.[34]havereportedthesurface
modification technologies, such as ion implantation [35,36], ion beam-assisted deposition [37],
electrochemicaltechniques[38],ionexchange[39],sol-gel[40],sputtering[41,42],plasmaspray[43]
andchemicalvapordeposition[44]. However,micro-arcoxidation(MAO)isnotfullydescribed.
MAO,whichcanproduceporous,adhesiveandbioactivecoatingsforimplantation,hasaroused
considerable attention [45–47]. On the one hand, a porous bioactive calcium phosphate-based
compositelayercanbedepositedonTi-basedimplantsurfacesaccordingtotheselectedelectrolyte,
whichwouldenhancethebiocompatibility[48,49]andthebondingstrengthofthecoatedlayer[50].
Ontheotherhand,antibacterialmetalelementscanbeincorporatedintoimplantsurfacestoinhibit
initialadhesionofbacteriaandpreventpost-surgerycomplications,thusenhancingtheantibacterial
property[34]. Furthermore,thecontentofbioactiveelementsandantibacterialmetalelementsonthe
MAOcoatingsurfacecanbetunedbycontrollingvoltage,electrolytecomponentsandMAOtime[51].
Toourbestknowledge,thisspecifictopichasnotbeenreviewedintherecentliterature. Theaimofthe
reviewistocollectandcomparetherecentscientificpapersconcerningsurfacemodificationofTiwith
theincorporationofantibacterialmetalelements(e.g.,Ag,Cu,Zn)toendowantibacterialproperties
byMAO.Thereviewfocusesmainlyontheeffectsofthecompositionandconcentrationofelectrolyte
andprocessparameters(voltage,currentdensity,oxidationtime)onthemorphologicalcharacteristics,
antibacterialabilityandbiocompatibilityofMAOcoating. MAOassistedbymagnetronsputtering
(MS)toachieveasuperiorantibacterialpropertyandbiocompatibilityisalsodiscussed,followingthe
forecastofthedevelopmenttrendofMAOinthefuture.
2. Micro-ArcOxidationMethod
MAO, which is also referred to as plasma electrolyte oxidation (PEO), is a high voltage
plasma-assisted anodic oxidation process. MAO is a relatively convenient technique for forming
firmlyadherentoxideceramiclayersonthesurfacesofvalvemetal,suchasTi,Al,Mg,Zr,Taand
theiralloys. AsshowninFigure1,theMAOprocesswascontrolledbyanMAOpowersupply. Before
MAO,thenon-workingsideofthespecimenwasconnectedwithacopperconductorandcoatedwith
acrylateadhesive. DuringtheMAOprocess,thespecimenwastreatedastheanode,andthestainless
steel electrolytic bath was regarded as the cathode to fill with electrolyte. The stirrer was applied
tokeeptheelectrolyteuniform,andthecirculatingwatertreatmentsystemwasusedtosustainthe
temperatureoftheelectrolytebelow30◦C.
TheprincipleofMAOisexplainedinFigure2. AttheinitialstageofMAOtreatment,assoonas
thespecimenisexposedtotheelectrolyte,thevoltageincreasesrapidlyandlinearlywithtime,andan
anodicbarrierfilm,alsocalledapassivatingfilm(Figure2a),isinitiallyformedonthesurfaceofthe
specimen. Soonafterwards,withtheincreaseofvoltage,sometinyoxygenbubblescanbeobserved,
andaporousinsulatingoxidelayer,whichusuallygrowsunderconditionsofdielectricbreakdown,
formsonthesurfaceofthespecimen[52]. Atthisstage,thevoltageandcurrentflowfollowFaraday’s
law,whichcorrespondstotheconventionalanodizingstage(Figure2b). Whentheappliedvoltageon
thespecimensurpassesthedielectricbreakdownoftheinsulatingoxidecoating,dielectricbreakdown
takesplace,leadingtotheformationofsparkdischarges(Figure2c).Thecurrentflowonlyconcentrates
onregionsofbreakdown,andtheCaandPelementsfromtheelectrolyteandtheotherelementsfrom
thespecimenenterintoregionsofbreakdownbydiffusionandelectrophoresisatintenselocalhigh
temperatures, resulting in localized forming and thickening of the porous structure oxide coating.
The discharge sparks gradually grow bigger, and the micro-arcs’ discharges are transformed into
powerfularcdischarges(Figure2d). Whenthenewgeneratedoxidecoating(Figure2e)iscapableof
resistingcurrentflow,theotherregionsarevulnerabletobreakdownduetothesmallerresistance,and
Coatings 2017, 7, 45   3 of 22 
CoCatoiantginsg2s0 21071,77,, 74,5 45   3 o3f 2o2f 22
generated oxide coating (Figure 2e) is capable of resisting current flow, the other regions are 
generated oxide coating (Figure 2e) is capable of resisting current flow, the other regions are 
vulnerable to breakdown due to the smaller resistance, and finally, the chemical reaction interface 
finvaulllny,etrhaeblceh teom birceaalkrdeaocwtino nduinet etorf tahcee swmoaulllderm reosvisetatonwcea, radnsdt hfienaelnlyti,r ethseu crhfaecmei[c5a3l] r.eBarcetaioknd oinwtenrfoafceth e
would move towards the entire surface [53]. Breakdown of the coating occurs at a vulnerable spot of 
cowatoinugldo mccouvres taotwaavrdusl ntherea ebnlteirsep soutrofafcteh [e5g3]r.o Bwreinagkdooxwidne ofif ltmhe. Mcoaetainnwg ohcilceu,rtsh aet nae vwulpnoerraobulse sstproutc touf re
the growing oxide film. Meanwhile, the new porous structure oxide coating might also be formed 
oxtihdee gcrooawtiinngg moxiigdhet faillmso. Mbeefaonrwmheidle,a tnhde tnheiwck penoerodubs ysttrhuecteumrei sosxioidneo cfogaatisngd umeigtohti natleson sbeel ofocramlhedig h
and thickened by the emission of gas due to intense local high temperatures at the sites of 
teamnpde rtahtiuckreesneadt thbye stihtees eomfbisrseiaoknd oofw gna[s5 4d]u.eW tioth itnhteenpsreo llooncagli nhgigohf otxeimdpateiroantutriems ea,tl atrhgee dsiitsecsh aorfg e
breakdown [54]. With the prolonging of oxidation time, large discharge channels with intense 
chbarnenaekldsowwinth [5in4t]e. nWseitshp atrhkei npgroalnodnggiansgb uofb bolxeisdwatoiounld teimmee,r glaeragte thdeisscuhrafragcee ,clehaadninneglst owtihthe fionrtmenasteio n
sparking and gas bubbles would emerge at the surface, leading to the formation of larger protruding 
sparking and gas bubbles would emerge at the surface, leading to the formation of larger protruding 
oflargerprotrudingpores,evenaspongyinterconnectedmicrostructure(Figure2f). Thecontinuous
pores, even a spongy interconnected microstructure (Figure 2f). The continuous formation and 
pores, even a spongy interconnected microstructure (Figure 2f). The continuous formation and 
forbmreaatkiodnowannd ofb rtheaek odxoidwen cooafttihneg o(Fxiigduerceo 2agt)i ncgau(Fseigs uthree 2pgo)tecnatuiasle tsot hfleucptoutaetne.t iBaolttho tflhuec dtuisastoel.uBtiootnh otfh e
breakdown of the oxide coating (Figure 2g) causes the potential to fluctuate. Both the dissolution of 
disthsoel buatisoen moaftethriealb aansed mgaastiefirciaatlioann dofg tahsei fiecleacttiroonlyotfe tehneaeblleec tthroe lfyotremeantaiobnle otfh tehefo promroautiso nceorafmthiec opxoirdoeu s
the base material and gasification of the electrolyte enable the formation of the porous ceramic oxide 
cercaomatiicngo x(Fidigeucroea 2thin).g  (Figure2h).
coating (Figure 2h). 

Figure 1. Schematic diagram of the micro‐arc oxidation (MAO) experimental device. 
FiFgiugurere1 1..S Scchheemmaattiicc ddiiaaggrraamm ooff tthhee mmiiccrroo‐-aarrcc ooxxiiddaattiioonn ((MMAAOO) )eexxppereirmimenetnatla dledveivceic. e.

Figure 2. Schematic of the formation process of MAO porous coating. The passivating film (a), and 
FiFgiugruere2 .2. SScchheemmaattiicc ooff tthhee foformrmataiotino nprporcoecsse sosf oMfAMOA pOorpoourso cuosatcinoga.t iTnhge. pTahsseivpaatisnsgiv failtmin g(afi), lamnd( a),
porous insulating oxide film (b) is initially formed. Then under the action of the spark discharges (c) 
anpdorpoourso uinssiunlsautilnagti noxgidoxe ifdilemfi (lmb) (isb )inisitiianliltyia flolyrmfoerdm. Tehde.nT huenndeurn tdheer atchtieoanc otifo tnheo fspthaerks pdaisrckhdarisgcehsa (rcg) es
and, powerful arc discharges (d), the new generated oxide coating (e) is formed and thicken. Finally, 
and, powerful arc discharges (d), the new generated oxide coating (e) is formed and thicken. Finally, 
(c)and,powerfularcdischarges(d),thenewgeneratedoxidecoating(e)isformedandthicken.Finally,
the porous ceramic oxide coating is formed (h) with the continuous formation and breakdown of the 
the porous ceramic oxide coating is formed (h) with the continuous formation and breakdown of the 
theoxpiodreo cuosactienrga m(gi)c aot xthidee lacrogaet idnigscihsafrogrem cehdan(hne)lws (ifth). thecontinuousformationandbreakdownofthe
oxide coating (g) at the large discharge channels (f). 
oxidecoating(g)atthelargedischargechannels(f).
The  composition  of  the  MAO  electrolytes  has  a  dramatic  effect  on  the  morphological 
The  composition  of  the  MAO  electrolytes  has  a  dramatic  effect  on  the  morphological 
chaTrhacetecriosmticpso, ssuitciohn aso tfhet hpeoroMsiAtyO anedl etchtircoklnyetesss ohf aMsAaO dcroaamtinagtisc. Heeffneccet, tohne ptrhoepemr oserlpehctoiolong oicfa l
characteristics, such as the porosity and thickness of MAO coatings. Hence, the proper selection of 
chtahrea cetleercitsrtoiclyst,es ucochmpasostihtieonp oisr oismitpyeraantdiveth tioc konbetsasino fsaMtisAfaOctocroya tpinegrfso.rmHaennccee. ,Gtheeneprraollpye, rdsifefleercetniot n
the electrolyte composition is imperative to obtain satisfactory performance. Generally, different 
ofatnhteibealectcetrrioally mteectaolm elpeomseitniotsn misixiemdp weritahti cvaelctioumob atacientastae t(iCsfAac) taonrdy pgleyrcfeorrompahnocspe.hGateen deirsaoldlyiu,mdi f(fGerPe)n t
antibacterial metal elements mixed with calcium acetate (CA) and glycerophosphate disodium (GP) 
are used as the base electrolyte in the MAO process to enhance the antibacterial property of the 
anatrieb aucsteedri aalsm theeta blaeslee meleencttrsomlyitxee idn wthiteh McaAlcOiu pmroacceestsa tteo (eCnAha)nacned tghley caenrtoibpahcotesrpiahla pterodpiseortdyi uomf th(Ge P)
implant materials. The characteristics of the fabricated antibacterial coating prepared by MAO 
ariemupsleadnat smthaetebraiasles.e lTehcter oclhyateraicntethriestMicsA Oof ptrhoec efsasbtroiceantehda nacnetitbhaecatenrtiiabla cctoeartiianlgp rporpeepratyreodf tbhye iMmApOla nt
techniques are briefly summarized in Table 1, and the details are reviewed according to the difference 
mtaetcehrinailqsu.eTsh aerec hbarireafclyte sruismticmsaorfiztehde ifna bTraibclaet e1d, aanndt itbhaec dteertiaaillsc aoraet irnegvipewreepda arecdcobrdyiMngA toO thteec dhinffieqrueenscea re
of the doping method of the antibacterial metal elements. 
broief flthyes duompminagr mizeedthiondT oafb tlhee1 a,natnibdatchteeridael tmaielstaal reelermeveinetws.e daccordingtothedifferenceofthedoping

m ethodoftheantibacterialmetalelements.
Coatings2017,7,45 4of22
Table1.SummaryofthecharacteristicsofantibacterialcoatingbyMAO.
ElectricalParameter Release
SurfaceContent
TiAlloy Electrolyte Vo(lVta)ge (ADC·uednrmrsei−ntyt2) OTxiimdeat(iso)n Surfa(PceorTeoSpiozger)aphy XRDPDhaesteected ofA(wg/tC%u)/Zn AAmg(/poCupunb/t)Zonf BTaecstteerdia Biocompatibility References
0.02MCa-GP,0.15MCA, Porousstructures(<3µm) Ti,rutileand
Ti-6Al-7Nb and3.0g·L−1AgNPs <250 20 300 withAgNPof37nm anatase 0.03 – S.aureus – [55]
0.02MCa-GP,0.15MCA, Porousstructures(<5µm)
Ti-6Al-7Nb and3.0g·L−1AgNPs <250 20 0–300 withAgNPof7–25nm – – – – – [56]
0.02MCa-GP,0.15MCA, Human
Ti-6Al-7Nb and0.3g·L−1AgNPs 234±3 20 300 Porousstructures(<5µm) – – 12 S.aureus osteoblasticcell [38]
0.02MCa-GP,0.15MCA, Human
Ti-6Al-7Nb and3.0g·L−1AgNPs 237±2 20 300 Porousstructures(<5µm) – – 89 S.aureus osteoblasticcell [38]
2g·L−1NaOH,15g·L−1 Porousstructures(<5µm) Escherichia
Cp-Ti NaH2PO4and3.0g·L−1CuNPs – 20 300 withCuNPof60nm – – – S.acuolrie,us – [57]
Human
0.04Mβ-GP,0.4MCAand Ti,rutile,and
Cp-Ti 250–350 – 180 Sphericalpores(<3µm) – – S.aureus osteosarcoma [58]
0.004MAgNO3 anatase (HOS)cell
Irregularandroughpores Rutile, Human
0.04Mβ-GP,0.4MCAand
Cp-Ti 0.004MAgNO3 420 – 180 withspahnedricflaalkpearticles α-βT-CCPa2aPn2dOH7,A 0.21–0.45 – S.aureus os(tHeoOsSa)rccoemlla [58]
Irregularandroughpores Rutile, Human
0.04Mβ-GP,0.4MCAand
Cp-Ti 0.00006MAgNO3 420 – 180 withspahnedricflaalkpearticles α-βT-CCPa2aPn2dOH7,A 0.1 – S.aureus os(tHeoOsSa)rccoemlla [58]
Granular Ti,rutile,
Ti6Al4V β-GP,CAand0.1g·L−1AgNO3 400 – 300 morphologywithAgNPs anatase, 0.6 2500 E.coli – [59]
of20–30nm CaTiO3andHA
Ti,rutile,
Ti6Al4V β-GP,CAand0.4g·L−1AgNO3 400 – 300 wNiethedAleg-lNikPesmoofr2p0h–3o0longmy anatase, 2.1 8000 E.coli – [59]
CaTiO3andHA
Flake-likemorphology Ti,rutile,
Cp-Ti Na2HPO4,CA,and0.0025M 380 – 300 withregionalAgparticles anatase, 4.6 – E.coli, – [60]
CH3COOAg lessthan200nm CaTiO3andHA S.aureus
Highlyorderednanopores Newbornmouse
Cp-Ti 0.5–1.0g·L−1AgNO3 – 65 5–240 withAgNPsof10–30nm – – 200–450 S.aureus pre-osteoblast [61]
withinmicropits cells
Cp-Ti Ca7(.N6gO·3L)−21anNda31P.0Og4·,L9−.41gA·gLN−1O3 – 65 240 HwiigthhwlAyigtohrNidnPemrseoidcfrn1oa0pn–iot3s0ponrmes – – – S.aureus Nperwe-boocsertnlelosmbloaustse [61]
Coatings2017,7,45 5of22
Table1.Cont.
ElectricalParameter Release
SurfaceContent
TiAlloy Electrolyte Vo(lVta)ge (ADC·uednrmrsei−ntyt2) OTxiimdeat(iso)n Surfa(PceorTeoSpiozger)aphy XRDPDhaesteected ofA(wg/tC%u)/Zn AAmg(/poCupunb/t)Zonf BTaecstteerdia Biocompatibility References
Cp-Ti 1.0~8.0g·L−1Cu(NO3)2 – 65 240 Mewsoitphoirnems(i2c0ro–4p0itsnm) Ti – – – Osteoblastcells [62]
Cp-Ti 3.8~17.0.6gg·L·L−−11CNua(N3POO34)2and – 65 240 Mewsoitphoirnems(i2c0ro–4p0itsnm) Ti – – – Osteoblastcells [62]
Microporesorcrater
0.05Mβ-GP,0.1MCAand Human
Cp-Ti – 16.5 240 structures(3–5µm)with Tiandanatase 1.43 – S.aureus [63]
0.05M(CH3COO)2Cu nano-grainsof30–50nm osteosarcomacell
0.02Mβ-GP,0.2MCAand
Microporestructures Rutileand Mousefibroblast
Cp-Ti 0.00125~0.005MCu 450 – 90 0.67–1.93 2.8–60.2 S.aureus [64]
(1–4µm) anatase cell
(CH3COO)2
0.15MCa-GP,0.02MCAand Ti,rutileand E.coli,S.
Cp-Ti – 30 300 Porousstructures(<5µm) 8.7 – Osteoblastcells [65]
0.06MZA anatase aureus
Ratbone
0.05Mβ-GP,0.1MCAand Microporousstructures E.coli,S.
Cp-Ti – 16.5 240 – 4.6–9.3 1000–3620 mesenchymal [10]
0.02~0.06ZA (<5µm) aureus
stemcells
0.02Mβ-GP,0.1MCA,0.1M Microporousstructures Anataseand 1.06–1.42(Ag),
Cp-Ti ZA,and6g·L−1AgNPs 390 – 30–90 (1–4µm) rutile 22.19–26.93(Zn) – S.aureus – [66]
Microporousstructures
0.02Mβ-GP,0.1MCA,0.1M Anatase,rutile 1.56(Ag),29.38 684(Ag),
Cp-Ti ZA,and6g·L−1AgNPs 390 – 120 (1–4µm)withAgNPsof andZnO (Zn) 6880(Zn) S.aureus – [66]
5–10nm
Microporousstructures Anatase,rutile,
0.02Mβ-GP,0.1MCA,0.1M 1.58(Ag),31.27
Cp-Ti ZA,and6g·L−1AgNPs 390 – 240 (1–4µm)with ZnOand (Zn) – S.aureus – [66]
somedeposits Zn2TiO4
Notes:GP:Glycerophosphate;CA:Calciumacetate;α-TCP:Ca3(PO4)2;HA:Ca10(PO4)6(OH)2.
Coatings2017,7,45 6of22
2.1. IntroductionofMetalNanoparticlesintoMAOElectrolyte
NPspresentagreaterchemicalactivitythanthebulkmaterialfortheadvantageoftheirlarger
ratioofCsouatrinfgasc 2e01a7,r 7e,a 45t o  volume[67]. AgNPsarethemostpopularinorganicantimicrobialN6P osf 2[26 8]and
canbeincorporatedintoimplantsbydirectdispersionofsolidAgNPsintheelectrolyteduringMAO.
Theant2i.b1.a Icntterordiaulctmione cofh Manetiaslm Nsanaosparretipcloesr tinetdo MarAeOth EaletctAroglytNe Pscandirectlyinteractwiththemicrobial
cells,leadinNgPtso ptrheseenint car geraesaeteor fchmememicablr aacntieviptye rthmane athbeil bituylk[ 6m9a],tetrhiael dfoerg trhaed aadtviaonntaogfe loipf tohpeiorl lyasragecrc haride
molecurlaetsiow ofi tshurtfhacee faorrema taot ivoonlumofe p[6i7ts]. aAngd NgPas parse itnhet hmeosbta pcotperuilaalr imnoermgabnrica nanetiamnidcrotbhieal dNaPms a[6g8e] of the
bacteriaalndo ucatner bme ienmcorbproarnaeted[7 i0n,t7o1 i]m.pAlagntNs bPys dciarencta dlsisopepresinoent oraf tseoltidh eAbg aNcPtes riina tlhcee ellleectnrovleyltoe pdeurainngd  cause
MAO. The antibacterial mechanisms as reported are that Ag NPs can directly interact with the 
DNA damage and protein oxidation by producing secondary products, such as reactive oxygen
microbial  cells,  leading  to  the  increase  of  membrane  permeability  [69],  the  degradation  of 
species[72]. IthasalsobeendemonstratedthattheantimicrobialactivityofAgNPsishigherthanthat
lipopolysaccharide molecules with the formation of pits and gaps in the bacterial membrane and the 
ofAgion[73]. TheuseofAgNPswillleadtoanti-bacterialeffectsmaximizedduetotheincreased
damage of the bacterial outer membrane [70,71]. Ag NPs can also penetrate the bacterial cell envelope 
numberasndo fcapuasret iDclNesAp dearmuangiet aanrde apr[o6t7e]i.n oxidation by producing secondary products, such as reactive 
Neocxuylgaene tspale.c[ie5s5 []7h2]a. vIte hsaus caclseos sbfeuelnly defambornicstartaetdedA thga-tb tehaer ainntgimTiicOrobciaola atcintivgistyw oift Ahgd NiffPesr iesn htigchoenrt entsof
2
AgNPsthbayn MthaAt Oof Aing tihoen e[7l3e]c. tTrohley utese, ionf cAlug dNinPsg wCiAll l,ecaadl ctoiu amntig‐blayccteerrioapl ehfofescptsh mataexi(mCiaz-eGd Pd)uew tiot hthAe gNPs.
increased numbers of particles per unit area [67]. 
ThemorphologyofmetallicAgNPsissimilartothatoftheparticlesintheelectrolyte[56]. Theaverage
sizeofAgNNPecsuwlaa est mal.e [a5s5u] hreadvet soubccees3s7fu±lly6 fanbmric.aUtendd Aegr‐tbheaerainsgs iTsitOan2 ccoeaotifngesle wctirtho pdhiffoerreesnits cdonutreinntgs MAO,
of Ag NPs by MAO in the electrolyte, including CA, calcium glycerophosphate (Ca‐GP) with Ag NPs. 
AgNPswerehomogeneouslyabsorbedontheporoussurface(Figure3a,d)[38],transmittedtothe
The morphology of metallic Ag NPs is similar to that of the particles in the electrolyte [56].  
innerpTohree wavaelrlasgoe fsitzhee otfi tAagn iNuPms woxasid meeaalsounregdt thoe bseh 3o7r ±t- c6i nrcmu.i Utpndatehr sth(eF iagssuisrtean3cbe, eo)f [e5l5ec,5tr6o]pohroreemsisb edded
in the ddeunrisneg oMxAidOe, lAagy eNrP(sF iwgeurree h3ocm,fo)g[e5n6e]o.usTlyh eabisnocrboerdp oorna ttihoen poorfoAusg suNrfPascei n(Ftighueret it3aan,di)u [m38]d, ioxide
(TiO )mtraantsrmixithteads tnoo theef fiencntero npotrhee wsaullrsf aocf ethme toitrapnhiuomlo ogxyidoef atlhoengc othaet isnhgosrt.‐cAirlcluoitf pthatehsla (yFiegrusrwe 3ebr,ee)p  orous
2
withwe[5l5l-,5d6e]fi onr eedmbpeodrdeesdr iann tghien dgeinnses iozxeidfer olamyear (fFeiwgurnea 3nco,fm) [5e6te].r Tshuep intcoor5pµormati[o3n8 o],f wAgh iNchPsi sint hthee typical
titanium dioxide (TiO2) matrix has no effect on the surface morphology of the coatings. All of the 
morphologyofMAO.Moreover,thecontentsofAgNPsonthesurfacewentupwiththeincreased
layers were porous with well‐defined pores ranging in size from a few nanometers up to 5 μm [38], 
amountofAgNPsintheelectrolyte. TheyfoundthatAgNPsdisplayedgoodantibacterialactivity
which is the typical morphology of MAO. Moreover, the contents of Ag NPs on the surface went up 
againstwmiteht hthiec iilnlicnre-aresesdis atamnotuSntta opfh Aylgo cNocPcsu isn atuhree eulsec(tSro.layuter.e uTsh)e,yo fnoeunodf tthhaet mAog sNtPpsr odbislpelmayaetdic gpooadth ogens
fororthaonptibeadcitceriimal palcatinvtitsy, waghaiinlests mhoetwhiicnilglinli‐trtelseisctayntto Stotaxpihcyiltoycotcocuas hauurmeusa n(So. asutereoubs)l,a ostniec ocfe ltlheli nmeowst iththe
adjustmperonbtleomfathtice cpoanthtoegnetnos ffAorg oNrtPhospoendiTc iOimp/lAangts,c owahtiinleg sbhyowdiencgr eliatstlien gcytthoteocxoicnitcye ntot raa tihounmoafn theAg
2
NPsinothsteeoeblleacsttirco cleyltle li[n7e4 w].itHh othwe eavdjeurs,ttmheenct ooaf tthine gcsonpterenpt oafr eAdg bNyPst hoins TmiOe2t/hAogd coaarteinegx bpye dnescirveeasdinuge  tothe
the concentration of the Ag NPs in the electrolyte [74]. However, the coatings prepared by this 
highpriceofAgNPs.
method are expensive due to the high price of Ag NPs. 

Figure 3. Low and high magnification surface SEM images (a,d); bright field TEM images (b,e), and 
Figure 3. Low and high magnification surface SEM images (a,d); bright field TEM images (b,e),
cross‐sectional SEM images (c,f) of the bactericidal coatings showing that the Ag NPs are distributed 
andcross-sectionalSEMimages(c,f)ofthebactericidalcoatingsshowingthattheAgNPsaredistributed
on the porous surface, in the micropores or embedded in the dense oxide layer. (Reprinted with 
on theppeormroiusssiosnu frrfoamc e[3,8i,n56t]h; Ceompyircirgohpt 2o0r1e2s Eolsreveimerb, Cedopdyerdighint 2t0h1e1 Edlesenvsieer.o) x ide layer. (Reprinted with
permissionfrom[38,56];Copyright2012Elsevier,Copyright2011Elsevier.)
Coatings2017,7,45 7of22
Cu can inactivate the central catabolic and biosynthetic pathways, namely catalytic clusters
of dehydratases [75], which endows Cu with strong antibacterial properties. Yao et al. [57] have
Coatings 2017, 7, 45   7 of 22 
preparedCu-dopedTiO coatingsbyMAOintheworkingelectrolytecontainingNaOH,NaH PO
2 2 4
and3g·L−1CCuu caNn Pinsa.cTtihvaeter etshue lctesnotrfatl ocpat-asbuorlfica caendm boiorspyhnothloetgici epsarthewveaaysle, dnatmhealty CcautaNlyPtisc cdluissttreirbs uotfe dboth
ontheCdue-hdyodpraetdasecso a[7ti5n],g wsuhircfha ceenadnowdsi nCsuid ewitthhe sptroornegs (aFnitgibuarcete4riaa)l. pTrhoepesritziees.o fYmaoo estt oalf. t[h5e7]N hPasvew  asless
than60pnremp.arHedi gChu-‐rdeospoeldu tTiioOn2 cXoPatSinsgps ebcyt rMaAinOd iinc athtee dwothrkaitngC eulemctraoilnyltye ceoxnitsatiendingin NtahOeHC, uN2a+H(2fPrOo4m  CuO)
and 3 g∙L−1 Cu NPs. The results of top‐surface morphologies revealed that Cu NPs distributed both 
state(Figure4b). Cu-dopedTiO coatingsshowedexcellentantibacterialpropertiesattributedtothe
2
on the Cu‐doped coating surface and inside the pores (Figure 4a). The size of most of the NPs was 
incorporationofCuNPswithahighsurfaceareatovolumeratio,whichwoulddirectlykillbacteria
less than 60 nm. High‐resolution XPS spectra indicated that Cu mainly existed in the Cu2+ (from CuO) 
andreleasecopperions. AsimilarmechanismwasreportedbyRaffietal.[76],inwhichthereason
state (Figure 4b). Cu‐doped TiO2 coatings showed excellent antibacterial properties attributed to the 
fortheainnctoibrpaocrtaetriioanl opfr Coup eNrPtys wofithC ua hNigPhs suwrafascem aareina ltyo avosclurmibee drattioo,t whehiachd hweosuioldn doirfeCctluy NkilPl sbaocntetroiab acteria
surfacesa,nnda rmeleealsye dcoirpepcetr cioonnst.a Act s-kimilillianr gm,escihnacneisCmu wNasP rseaproerteodp pbyo sRiateffliy etc ahla. r[7g6e]d, inw withhicrhe sthpee rcetatsoonb acteria.
Whenthfoery theen caontuibnatcetrereiaalc phrooptheretry, othf Ceure NdPusc wtiaosn mraeiancltyi oasncrwibieldl otoc cthuer aadthtehseiobna ocft Cerui aNlPcse ollnwtoa blal,ctreersiau ltingin
surfaces, namely direct contact‐killing, since Cu NPs are oppositely charged with respect to bacteria. 
theformationofcavities/pits. ThoughUsmanetal.[77]havealsotestifiedtotheantimicrobialand
When they encounter each other, the reduction reaction will occur at the bacterial cell wall, resulting 
antifungalactivitiesofCu-chitosanNPsagainstmethicillin-resistantS.aureus,B.subtilis,C.albicans,
in the formation of cavities/pits. Though Usman et al. [77] have also testified to the antimicrobial and 
P.aerugiannotsifauanngdal Sacatlimviotineesl loaf cChuo‐lcehriateossuains ,NPPasp aegaeitnastl .m[7e8th]ichiallvine‐rceosinstfiarnmt Se.d autrheauts,t Bh.e suabnttiliibs,a Cct. earlbiaiclaansc,t ivityof
CuNPsPw. aaesrusgiignnosiafi acnadn tSlaylmwoenaelklae crhtohlearanestuhisa,t PoafpAe egt aNl.P [7s8.]M haovree ocovnefri,rmCeudN thPast twhee arentribaapcitderliyalo axcitdiviiztye dwhen
exposedoft oCua iNrP[7s 9w],alsi msigintiinfigcatnhtleyi rwaepakpelri ctahtaino nth.at of Ag NPs. Moreover, Cu NPs were rapidly oxidized 
when exposed to air [79], limiting their application. 
WiththeconsiderationofthebaddispersionofNPsinsolutionandtheweakadhesionofNPsonto
With the consideration of the bad dispersion of NPs in solution and the weak adhesion of NPs 
thecoatingsurface,onlyseveralvaluablestudieshavebeendocumentedinthefieldofantibacterial
onto the coating surface, only several valuable studies have been documented in the field of 
NPsbyMAOsofar.
antibacterial NPs by MAO so far. 

Figure 4. SEM micrographs of Cu‐doped coatings revealing the presence of Cu nanoparticles on the 
Figure4.SEMmicrographsofCu-dopedcoatingsrevealingthepresenceofCunanoparticlesonthe
surface and inside the pores (a); high‐resolution XPS spectra of Cu 2p in TiO2 coating indicated that 
surfaceandinsidethepores(a);high-resolutionXPSspectraofCu2pinTiO coatingindicatedthatCu
Cu mainly exists in the CuO state (b). (Reprinted with permission from [57]; Cop2yright 2014 Elsevier.) 
mainlyexistsintheCuOstate(b).(Reprintedwithpermissionfrom[57];Copyright2014Elsevier.)
2.2. Introduction of Metallic Compounds into MAO Electrolyte 
2.2. IntroducFtoiorn thoef MbioemtaeldliiccaCl oimndpuosutrnyd, siti nseteomMs AtoO beE alenc tartotrlayctteive strategy to endow implants’ surfaces 
with metallic coatings. Recent studies [45,80,81] have shown that biological or antibacterial elements 
Forthebiomedicalindustry,itseemstobeanattractivestrategytoendowimplants’surfaceswith
can be incorporated into the coating during the MAO process by the introduction of metallic 
metalliccoatings. Recentstudies[45,80,81]haveshownthatbiologicalorantibacterialelementscan
compound into the MAO electrolyte. Ag‐containing coatings have occupied the largest share of the 
beincorgploobraalt aendtiibnatcotertihael mcoaarkteint. gduringtheMAOprocessbytheintroductionofmetalliccompound
into the MASoOnge elet catl.r o[5l8y]t he.aveA ign-ccoorpnotraaitnedin Aggc ionatot iTnig ismphlaavntes obyc cMuApiOe din t0h.0e4 lMar βg‐egsltycsehroaprheoospfhtahtee  global
antibactdeirsioadliumma sraklte pt.entahydrate (β‐GP), 0.4 M CA and silver nitrate (AgNO3) or silver acetate (CH3COOAg) 
Sonelgecetrtoalyl.ti[c5 s8o]luhtaiovne aitn ac ofirxpeodr aaptepdlieAd gvoilntatogeT riainmgep flraonmts 2b50y tMo A45O0 Vin. T0h.0e 4adMdiβtio-gn loyfc eArgoNpOh3o sphate
reduced the required voltage for the formation of calcium phosphate compounds from 450 down to 
disodiumsaltpentahydrate(β-GP),0.4MCAandsilvernitrate(AgNO )orsilveracetate(CH COOAg)
400 V [82,83]. When the concentration of AgNO3 was fixed at 0.004 M, the 3oxidized layer was a porou3s 
electrolyticsolutionatafixedappliedvoltagerangefrom250to450V.TheadditionofAgNO reduced
microstructure with spherical pores at voltages below 350 V (Figure 5a), while the coating surfa3ce 
therequbierecadmveo rlotauggeh faonrdt hceovfeorremd awtiiothn sopfhcearilccailu pmarptihcloess pahndat eflackoems paboouvne d3s80fr Vom (F4ig5u0red 5obw) ndutoe t4o0 0thVe [82,83].
Whentheexicsotenncceen otfr caatlicoiunmo fpAhogsNphOatew coamspfioxuenddas t(F0i.g0u0r4e M5c,).t hMeAoOx icdoiaztiendgsla cyoenrtawinainsga 0p.2o1r–o0u.4s5 mwti c%ro osft ructure
3
Ag were cytotoxic to human osteosarcoma MG63 cells, while Chen et al. [84] reported that 2.05 wt % 
withsphericalporesatvoltagesbelow350V(Figure5a),whilethecoatingsurfacebecameroughand
covered withsphericalparticlesandflakesabove380V(Figure5b)duetotheexistenceofcalcium
phosphatecompounds(Figure5c). MAOcoatingscontaining0.21–0.45wt%ofAgwerecytotoxic
Coatings2017,7,45 8of22
to human osteosarcoma MG63 cells, while Chen et al. [84] reported that 2.05 wt % Ag displayed
Coatings 2017, 7, 45   8 of 22 
noosteoblastcytotoxicity. Thecomparisonresultsindicatedthatthecellproliferationnotonlyhas
arelatioAngt oditshpelaycoedn tneon otsotefoAblga,stb cuyttoatlosxoicditye.p Tehned csoomnpaortihsoenr rfeascutlotsr si,nsduiccahtedas ththate thsue rcfeallc perrooliufegrhatnioens softhe
not only has a relation to the content of Ag, but also depends on other factors, such as the surface 
coatings[85]. Therelativelysmoothcoatingoxidizedat400VwithalowercontentofAg(0.00006M
roughness of the coatings [85]. The relatively smooth coating oxidized at 400 V with a lower content 
AgNO )exhibitednocytotoxicitywithanunreducedantibacterialproperty.
3of Ag (0.00006 M AgNO3) exhibited no cytotoxicity with an unreduced antibacterial property. 

Figure 5. Surface morphologies of MAO samples obtained in electrolyte solution containing 0.004 M 
Figure5.SurfacemorphologiesofMAOsamplesobtainedinelectrolytesolutioncontaining0.004M
AgNO3 for 3 min at 350 V (a), and 400 V (b); (c) the cross‐section view of the specimen in (b) [58]. 
AgNO3for3minat350V(a),and400V(b);(c)thecross-sectionviewofthespecimenin(b)[58].
(Reprinted with permission from [58], Copyright 2009 John Wiley & Sons.) 
(Reprintedwithpermissionfrom[58],Copyright2009JohnWiley&Sons.)
Muhaffel et al. [59] have also prepared novel multi‐layer coatings composed mainly of inner 
TiO2 layers and outer HA (HA (Ca10(PO4)6(OH)2)) layers via MAO in β‐GP and CA electrolyte with 
Muhaffeletal.[59]havealsopreparednovelmulti-layercoatingscomposedmainlyofinnerTiO
different addition levels of AgNO3 (0.1 g∙L−1 and 0.4 g∙L−1). Unlike the general surface characteristics  2
layersandouterHA(HA(Ca (PO ) (OH) ))layersviaMAOinβ-GPandCAelectrolytewithdifferent
of MAO coatings, micro‐1p0ores w4e6re hard2ly identified owing to the existence of outer granular HA on 
additionMlAevOe‐l0s (oFfigAugreN 6Oa)3, M(0A.1Og‐·0L.1− (1Faignudre0 6.4b)g o·Lr −n1e)e.dUle‐nlilkike eHtAhe lagyeenrse roanl sMuArfOac‐0e.4c h(Fairgaucrtee r6ics)t. iTcsheo fMAO
coatingsa,dmdiitciorno -opfo AregsNwO3e rceanh sapredeldy uidpe tnhtei fiMeAdOo wprioncgestso atth heigehxeirs tceunrcreenot fdoenustietyr glervaenlsu, leanrhHanAce othneM  AO-0
(Figurec6ray)s,taMlliAniOty -o0f. 1th(eF HigAu lraeye6rb a)nodr pnreeceidpiltea-tiloikne ofH AAg NlaPyse orns othne MHAA lOay-e0r..4 C(oFmigpuarreed6 tco) a.mTohrephaoduds itionof
HA, the crystalline HA exhibits better bioactivity in SBF (simulated body fluid) and biointegration 
AgNO canspeeduptheMAOprocessathighercurrentdensitylevels,enhancethecrystallinityofthe
3
[86]. Ag NPs with a size of 20–30 nm were deposited on the HA layer of MAO‐0.1 (Figure 6e) and 
HAlayerandprecipitationofAgNPsontheHAlayer. ComparedtoamorphousHA,thecrystalline
MAO‐0.4 (Figure 6f). The rapid release of Ag+ ions at the initial periods and the subsequent slower 
HAexhrieblietasseb ceotntetrribbuioteadc ttoiv loitnyg‐itnerSmB aFnt(isbiamctuerliaatle adctibvoitdy yocflcuurirded).a Mndoreboivoeinr, tceugmrautlaiotinve[ r8e6le].asAe gofN APg+s witha
sizeof2io0n–s3 (02.n5 mppmw eforre MdAepOo‐0s.i1t eanddo 8n ptphme fHorA MAlaOy‐e0r.4o) fwMas AloOw-e0r .t1ha(nF itgheu troexi6ce c)oanncedntMratAioOn (-100.4 pp(Fmig) ure6f).
The rapoidf Areg lefoars ehuomfaAn gc+elliso [n8s7]a. tOtvheerailnl ictoinaslidpeerraitoiodns, 0a.n1 dg∙Lth−1e AsguNbOse3 qauppeenatresdlo two eerxhriebliet assuefficcoiennttr ibuted
tolong-atnetrimbacatenrtiiabl aacctteivriitayl aancdti vniot yrioskc coufr rceydto.toMxiocirtye,o vwehri,lec upmresuelravtiinvge trheel esatrsuectoufraAl gch+airoanctser(i2st.i5csp. pmfor
MAO-0.M1uahnadffe8l petp aml. [f6o0r] MhaAveO fu-0rt.4h)erw reapsolrotewde ar mthualnti‐ltahyeert ocxoaictincog nccoennsitsrtaintgio onf (s1u0bnpaptamnt) ToifOA2 lgayfeorrs human
cells[87a]n.dO uvpeprearl lbcioo‐nmsiimdeetrica tpiroenci,p0it.1atigo·nL i−n1 dAisogdNiuOm haypdproegaerne dphtoospehxahtieb (iNtas2uHffiPOci4e),n CtAa natnidb a0c.0t0e2r5i aMl activity
CH3COOAg electrolyte at 380 V. Incorporation o3f 4.6 wt % Ag into the multi‐layer led to superior 
andnoarnistkibaocftecryiatlo etfofixciiecnitcyy, awgahinilset Epsrcehseerircvhiian. gcoltih (Ee. sctorliu) catnudr Sa.l acuhreaursa wctheirlei sctoincsse.rMvinugh tahfef eblioemtiaml.et[i6c 0]have
furtherarpeaptoitret perdecaipmitautilotni-. lTahyee rmcuoltailtaiynegr ccoonnsissitsetdin ogf tohfreseu labynearst:a Ant thTiinO c2omlapyaecrt sTiaOn2d lauyeprp aebrovbei oth-em  imetic
precipitasutibosntrainte dshisoowdiniugm behttyerd sruobgsetnratpeh boosnpdhinagte, a( NmaiddHleP pOor)o,uCs Aanadn tdhic0k.0 T0i2O52 MlayCerH onC thOeO cAomgpealcetc trolyte
2 4 3
layer and a flake‐like top layer of biocompatible compound incorporated with regional Ag particles 
at 380 V. Incorporation of 4.6 wt % Ag into the multi-layer led to superior antibacterial efficiency
(Figure 7). 
againstEscherichia. coli(E.coli)andS.aureuswhileconservingthebiomimeticapatiteprecipitation.
Unlike the common surface micrometer‐sized pores of MAO coatings, highly ordered nanopores 
Themultilayerconsistedofthreelayers: AthincompactTiO layerabovethesubstrateshowingbetter
(Figure 8b,c) within micropits (Figure 8a) were successfully p2repared by Chang et al. [61] via a novel 
substratoeneb‐ostnepd ihniggh,‐acumrreidntd alneopdiozraotiuons (aHnCdAt)h aitc tkheT ciuOrr2enlat ydeenrsoitny otfh 6e5 cAo∙dmmp−2a icnt tlhaey AegrNaOn3d elaecfltraoklyet-el iketop
layerofrbaniogceo fmropma t0i.b5–le16c ogm∙L−p1. oTuhne dnainncooporpreo draiatmedetwer itdhecrreegasieodn aalndA gthpe awratilcl ltehsic(kFniegsus rienc7r)e.ased with 
Unilnikcreeathsiengc oofm AmgNonO3s cuornfcaecnetrmatiiocnros m(Fiegtuerre- s8idz)e. dDupeo troe sthoe fcoMnAtroOllacbolea tdiinmgesn,shioignsh, ltyheo nrdaneorpedoronuasn opores
structure modulated osteoblast functions, accommodating various clinical needs [88–90]. Scratch 
(Figure8b,c)withinmicropits(Figure8a)weresuccessfullypreparedbyChangetal.[61]viaanovel
tests showed significantly enhanced bonding strength on the HCA treated sample (Figure 8e) in 
one-stephigh-currentanodization(HCA)atthecurrentdensityof65A·dm−2intheAgNO electrolyte
comparison with flaking on the anodized sample (Figure 8f), even at a small normal load. The 3TEM 
rangefraonmd X0R.5D– 1sh6ogw·eLd− t1h.atT Ahge wnaasn eompboerdeddedia imn ecrtyesrtadlleizcerde aTsieOd2 iann tdhet hfoermw oafl lcrtyhsitcakllnizeesds AingOcr eNaPsse dwith
increasinwgitho fdAiagmNetOer3s cboentwceenetnr a1t0io nnms( Faingdu r3e0 8ndm).. DTuhee tcorytshteallciozendt roTlilOa2b laelsdo imcoenntrsiibountesd, thtoe ngoaondo porous
structure modulated osteoblast functions, accommodating various clinical needs [88–90]. Scratch

tests showed significantly enhanced bonding strength on the HCA treated sample (Figure 8e) in
comparisonwithflakingontheanodizedsample(Figure8f),evenatasmallnormalload. TheTEM
andXRDshowedthatAgwasembeddedincrystallizedTiO intheformofcrystallizedAgONPswith
2
Coatings2017,7,45 9of22
diametersbetween10nmand30nm. ThecrystallizedTiO alsocontributedtogoodbioactivity[49].
2
Coatings 2017, 7, 45   9 of 22 
Gaoetal.[91]reportedthatseveralppbofAgreleasedquantitywerecapableofkillingallthebacteria.
Coatings 2017, 7, 45   9 of 22 
Thereforbeio,athcteivhitiyg [h49r]e. leGaasoe elte vale. l[o91f]A regp(o>rt2e0d0 tphpatb s)erveevraela plepdb sotfr oAngg raenletaismedic qroubaniatiltyp rwoepreer ctiaepsa,bbleu tofr esulted
inseverbCkeioiolacltaiiynncgttgsoi  v2ati0lotl1y 7xt h, [i74ce, 9 i4bt]5ay.  c .GtTearooia ie.m tT haple.rr oe[9fvo1e]r ert,eh tpheoec rhtyeigtdoh tc rhoealmet aspseeav lteeirvbaeli ll piotpfy bAa gon f( d>A2r0ge0 d rpeuplebca)es retedov xeqaiucleaidtnyt si,ttNyro wnageP raenO tciampaiancbrd9ol eobC fi o2aa2fl   (NO )
3 4 3 2
wereadkpdirleolidpnegirn atitlelo st,ht behu beta ercelteseurciltatre.o dTl hyinetr eseeftvooererie,n t hchyeitb hoititgothxhi rceeitlfyeoa. rsTemo l eiamvteipolr onofv oAef gthn (e>a 2nc0yo0t popcoporbme) spreatvotiebaailleictdey sr attrnaodinn rgee adxnuttceimen ttiocaxroincbdiitaytl,o   lower
thereleapbNsiroaoe3apPoceOtfrivt4Ai ieatgsyn, + d[b4 uw9Ct] ah.r (eGiNsleauOolpt3 e)er2dt e w sianele. r rs[eve9 vi1ane]dr gdere etcphdyo etroiinttreotdoax  nittchhtiitaeyb t. ea sTlceeotvce teirrmroiaalplyl rtpoaepv btbeio  lt oihitfney h A.ciygbt iotr ectolhemea spfeoadrt imbqiualtiatinoytn ai tnyod f w rneeadrneuo ccpeao ptroaexbsi lcteio to yaf,   
Nkceialrl3tiPaniOgn 4 a elaxl nttehdne t Cb aaanc(Ndte Otroia3 )l.2o T wwheeerrr eetf hoaerd erd,e etlhedae s ihnei tgoohf  t Arhegel+e  eawlsehec iltlereov pleyrl teoesf e tArovg ii n(n>gh2 it0bh0iet p irtph aben)  tfrioebrvamecataelteridioa nsl  tarobofi nlnigtay an.n otpimoriecsr otboi aal  
properties, but resulted in severe cytotoxicity. To improve the cytocompatibility and reduce toxicity, 
certain extent and to lower the release of Ag+ while preserving their antibacterial ability. 
Na3PO4 and Ca(NO3)2 were added into the electrolyte to inhibit the formation of nanopores to a 
certain extent and to lower the release of Ag+ while preserving their antibacterial ability. 

Figure 6. Low and high magnification SEM micrographs of MAO‐0 (a,d); MAO‐0.1 (b,e) a nd  
Figure 6. Low and high magnification SEM micrographs of MAO-0 (a,d); MAO-0.1 (b,e) and
FMigAuOre‐0 .64.  (Lc,of)w [ 59a]n. d(R hepigrhin tmeda gwniitfhic pateiromn isSsEioMn  fmroimcr o[5g9r]a,p Chosp oyfr igMhAt 2O0‐106  E(al,sde)v; ieMr.)A O‐0.1 (b,e) and  
MAO-0.4(c,f)[59].(Reprintedwithpermissionfrom[59],Copyright2016Elsevier.)  
MAO‐0.4 (c,f) [59]. (Reprinted with permission from [59], Copyright 2016 Elsevier.) 
Figure 6. Low and high magnification SEM micrographs of MAO‐0 (a,d); MAO‐0.1 (b,e) and  
MAO‐0.4 (c,f) [59]. (Reprinted with permission from [59], Copyright 2016 Elsevier.) 

Figure 7. Low magnification surface (a) and cross‐sectional SEM (b) micrographs for the MAO‐ Ag 
Fsaigmuprele s7 .o Lbotawin meda ginn iefilceacttrioonly steu rsfoalcuet i(oan)  aconndt acirnoisnsg‐s e0c.0ti0o2n5a Ml S ECMH3 C(bO) OmAicgr o(g1:r acpohmsp faocrt  tThieO M2 lAayOe‐rA; 2g:  
Figure7. Lowmagnificationsurface(a)andcross-sectionalSEM(b)micrographsforthe MAO-Ag
spaomropuless T oiObt2a lianyeedr ;i n3:  eblieocctoromlyptaet isbolleu ctioomn pcoounntadi ncionngt a0i.n0i0n2g5  lMay eCrH); 3(Cc)O hOigAhg m (1a:g cnoifmicpataicotn T siuOr2f alacye eSrE; M2:  
samplespFmoiogircbuorutoraegs i r7Tna.i peOLhdo2s wl iainny mdeeiracl; gae3tnc:e tibdfrii ocothaclytoaitmot ernpe gssaouitoirlbnuflaaetcl i ceAoo (ngma )ppc aaoornuntdintc adlceir scno ocsinosn‐tvgsaeeirnc0eti.din0o g0tnh 2laael5  ysSeuMErr)Mf;a (C cc(e)bH  [h)6 3im0gC]hi.O c (mrRoOeagpgArrnaigpinfhitc(es1ad tf: iowocrni ott hhsmu epr peMfaramcAcetiO sSsT‐EiAioMOgn   2 layer;
2:poroumsfsraoimTcmripoO l[ge62rs0a l]opa, bhyCtseoa iripnn;yde3rdi:icg abihntiet o ed2cl 0eto1hcm5tar tEop rllaeysgetteivibo isenloeral.lu)c  Atoiomgn pp acoortunictnaledins cicnoogvn e0tra.e0id0n2 it5nh egM sl uaCryHfae3crCe) O;[6(Oc0)]A. h(gRi g(e1ph: rcmionmtaegdpn awcifiti tTchai pOtei2or lmnayisseusri;ro f2na:  ceSEM
microgrfparopormhosu [s6i n0T]di,O Cic2o alpateyyrdeirg;t hh3t:a  b2t0ior1ec5og EmilospneavatiilebrAl.e) g copmaprtoiucnleds ccoonvtaeirneindgt lhaeyesru); r(fca) cheig[h6 0m]a.g(nRiefipcartiinotne sduwrfaicteh SpEeMrm  ission
micrographs indicated that regional Ag particles covered the surface [60]. (Reprinted with permission 
from[60],Copyright2015Elsevier.)
from [60], Copyright 2015 Elsevier.) 

Figure 8. Cont.   
Figure 8. Cont. 

Figure 8. Cont. 
Figure8.Cont.
Coatings2017,7,45 10of22
Coatings 2017, 7, 45   10 of 22 
Coatings 2017, 7, 45   10 of 22 

Figure 8. Low‐magnification (a) and high‐magnification (b,c) SEM images of the high‐current 
Figure 8. Low-magnification (a) and high-magnification (b,c) SEM images of the high-current
aannooddiizzaattiioonn ((HHCCAA))‐-ttrreeaatteedd ssaammppllee iinn AAggNNOO33 eelleeccttrroollyyttee;; ((dd)) vvaarriiaattiioonn ooff tthhee ppoorree ddiiaammeetteerrss  aanndd 
tthhiicckknneessss ooff tthhee HHCCAA‐-ttrreeaatteedd ssaammpplleess aass aa ffuunnccttiioonn ooff AAggNNOO3.. SSccrraattcchh SSEEMM iimmaaggeess ooff tthhee ssaammppllee 
Figure 8. Low‐magnification (a) and high‐magnification (b,c3) SEM images of the high‐current 
aannooddiizzeedd iinn tthhee eelleeccttrroollyyttee wwiitthh tthhee AAggNNOO3 ccoonncceennttrraattiioonn ooff 22 gg·∙LL−−11 ((ee)) aanndd tthhee ssaammppllee aannooddiizzeedd iinn 
anodization (HCA)‐treated sample in Ag3NO3 electrolyte; (d) variation of the pore diameters and 
eetthhyylleennee ggllyyccooll ssuupppplleemmeenntteedd wwiitthh 00..55 wwtt %% NNHH4FF,, 55..00 vvooll %% HH2OO aanndd 55..00 vvooll %% CCHH3OOHH aatt aann aapppplliieedd 
thickness of the HCA‐treated samples as a fun4ction of AgNO32. Scratch SEM images 3of the sample 
vvoollttaagganee ooodffi z33e00d VV in ff oothrr e11 .e.55l ehhc taraott lrryooteoo mmw itttehem mthppee eArraagttNuurOree3  ((cffo))n sschheoonwtwraeetddio tnthh oeef s s2iig ggnn∙Liiffi−i1cc (aaenn) ttallnyyd ee ntnhhheaa snnaccmeedpdl beb ooannndoddiinnizgge dsst trirnee nnggtthh 
oonn tthheeet HhHyCCleAAne‐-t tgrreleyaacttoeeldd s usspaammpleppmlleee n[[66te11d]]..  w((RRiteehpp 0rr.ii5nn wtteetdd % ww NiiHtthh4 Fpp, ee5rr.0mm viiosssls ii%oo nnH ff2rrOoo mamn d[[66 511.0]],, v CCoool %ppyy CrriHigg3hhOttH 22 00a11t 55a nEE allsspeepvvliiieeedrr.. )) 
voltage of 30 V for 1.5 h at room temperature (f) showed the significantly enhanced bonding strength 
LLiikkeewwonii sstheee,,  aaH CuuAnn‐iitqqreuuaeete ssdtt rrsuaumccttpuulerr ee[6 w1w].i it(tRhhe apar mimntiieccdrro ow-‐/i/thnn aapnneroom‐-mimssooiorrnpp fhhrooomlloo [gg61yy] ,cc Caaonnp yaarllissgooh tbb 2ee0 1oo5bb Ettalasieinnveieeddr. )ii nn CCuu((NNOO3))2 
3 2
eelleeccttrroollyyttee dduuee ttoo fifieelldd-‐aassssiisstteedd cchheemmiiccaall eettcchhiinngg ooff NNOO3−−. .HHuuaanngg eet taal.l .[[6622]] bbeelliieevveedd tthhaatt tthhee uussee ooff 
CCuu((NNOO3))2 Liiinnkssettweeaiasdde , ooaff u AAngigqNNuOeO s3t raaussct thtuhereee  wleelicetthcrt oarlo ymltyiectremo m‐a/ynaaaynv oao‐vmidoo3itdrhp ehthopeloo tgpeyon tcteiaannlt aciaylslto oc tbyoetx ooitcboittxayiicnoietfdyh  ieonaf C vhuye(maNvOeyt3 a)m2l ieotnasl. 
ions. eAleg3c+t2r owlyatse  edausei ltyo  ofixeildd‐iaz3sesdis tteod  hcihgehmeirc avl aeltecnhcineg ( oAf gN2+O),3 −w. Hhuicahn gw eitl la ll.e [a6d2 ] tboe lsieevveedre t hcaytt othteo xuiscei toyf  [61]. 
HAbigov+walweiCeovnaunets(srN.Ce,  AaOubsg32ii)+l+v2y  awiinnolaesxstnCie dteau aid(zCsN eiouldyfO2  +Ato3 ox)gi2nihNd ciiOgCazhen3u dean( rsNot ovttOh abhel3iee )gen2fh lceuececrrat tr(nvhAonaelgyloret2tneo+  c)xbme,ie wda( Ayifhz ugaie2crvd+th)oh., iwedwTr ihh tlhleiocelxhre iep adwsodiutizeltlelont dsltesi.aoae ldvfT c eEhtyroDete o sXctreoyevxatseoinucrtedilott yxscX y iocPtofoi Sftth yoceEx[oaDi6vcn1iyXfit]y . rm Ham[e6not1edaw]dl.   eXtvhPeaSrt, 
confirmed that toxic elements Cu were not incorporated into the coating surface, so copper will not 
toxicHeloewmeevnetrs, Cbiuvawleenrt eCnuo2t+ iinnc oCrup(oNrOat3e)2d cianntnootth beec ofuarttihnegr souxrifdaiczeed,s. oTchoe prpeesurlwts ilolfn EoDtdX iraencdtl yXPcoS ntact
directly contact the cells to produce cell toxicity. Cross‐sectional images (Figure 9) also indicated that 
thececlolnsftiormperdo dthuacte tocxeilcl etolexmiceintyts. CCuro wsse-rsee nctoito innaclorimpoargateesd( iFnitgou trhee 9c)oaatlisnogi snudrifcaacete, dsot hcoaptpheirg whliyll- noordt ered
hniagnhol-dyci‐hroearcndtlneyer celosdnw tnaecartne tohp‐eec crhepalelnsn ntdoei lpcsur owladreurtceoe  ctpheelelr tpsouexbnicsdittiryca.u tCelar,ora snsto‐ds eatchtcieoo nmsaupl biamscttargalaetyes ,e( Fraicngaudnr eab 9 e)c ooalbmssope iranvcdeti dclaabteyedet wrt hecaeat nn tbhee 
onbasneorphvioegrdhe lyab‐reortrawdyeesreeandn  dtnhatenh oen‐csahunabonspntorealrset e w.aeBrrreeas yipdse eraspn,etdnhd etichauedl adsriu ttbioos nttrhoaetf esP.u ObBs3et−rsaiidtnee, stha, netdhe elae  cactdormodlipytiatoecntf  ulaoryft ehPre Orcai34mn−   pbierno  vthede 
electrooblysetrev feudr tbheetrw iemenp rtohve enda tnhoep coerell ualrarra ypse rafnodr mthaen cseu b[9st2r,a9t3e.] . BMe4soidreeos,v ethre,  Aa dwdeitlilo‐dn eofifn PeOd 3m−  iicnr ot‐h/en ano‐
the cellular performance [92,93]. Moreover, A well-defined micro-/nano-morphology4 can also be
morpehleocltorgoyly tcea fnu ratlhseor  bime parcoqvueidr ethde b cyel tluhliasr H peCrAfo rtmecahnnceiq [9u2e,9 i3n] .e Mleocrteroovlyetre, As,  wsuecllh‐d aesf iCneud( NmOicr3o)2‐,/ nZann(oN‐O3)2, 
acquiredbythisHCAtechniqueinelectrolytes,suchasCu(NO ) ,Zn(NO ) ,(Na PO +Ca(NO ) +
(Na3PmOo4r +p hCoalo(NgyO c3a)n2  +al sNoa b2eS iaOcq3)u iarnedd  b(Ny tah3iPs OH4C +A A tegcNhnOiq3 u+e N ina e2lSeicOtr33o)2l, ywtehs,i cshuc wh3 ial2sl  Cexut(eNn3Od3 )i2t,4s Z anp(NplOic3a)2t, i3on2. 
Na SiO )and(Na PO +AgNO +Na SiO ),whichwillextenditsapplication.
2 (Na33PO4 + Ca(N3O3)42 + Na2SiO3)3 and (N2a3PO34 + AgNO3 + Na2SiO3), which will extend its application. 

Figure 9. (a) Low and (b,c) high magnification cross‐sectional SEM images of the high‐current 
FFiigguurree 99.. ((aa)) LLooww aanndd ((bb,,cc)) hhiigghh mmaaggnniiffiiccaattiioonn ccrroossss‐-sseeccttiioonnaall SSEEMM iimmaaggeess ooff tthhee hhiigghh‐-ccuurrrreenntt 
anodization (HCA)‐treated sample in Cu(NO3)2 electrolyte indicated the presence of highly‐ordered 
aannooddinizzaaanttoiioo‐cnnh a((HHnnCCeAlAs ))[‐-6tt2rr]ee. aa(Rtteeeddp rssiaanmmtedpp llwee iiitnnh  pCCeuurm((NNisOOsi33o))n22  eferlloeemccttr r[o6ol2ly]y,t teCe oiinpndydriicicgaahttete d2d0 t1thh6e eE plpsrreeevssieeenrn.c)c ee ooff hhiigghhllyy‐-oorrddeerreedd 
nnaannoo‐-cchhaannnneellss [[6622]].. ((RReepprriinntteedd wwiitthh ppeerrmmiissssiioonn ffrroomm [[6622]],, CCooppyyrriigghhtt 22001166 EEllsseevviieerr..)) 
As an essential trace element for living organisms, Cu participates in a variety of metabolic 
AAacsst ivaainnti eeesss, ssseeonn itttii aamll itgtrrhaatcc beee  eemlleeommreee nsnutt itffaoobrrl ell iifvvoiirnn aggn tooibrrgagcaatnneriisisamml assp,, pCCliuuc apptiaaornrtt iicccoiimppaapttaeersse diinn t oaa A vvgaa. rrZiieehttuyy  eoot ffa lmm. [ee6tt3aa] bboolliicc 
aaccttiivviipttirieeesps,,a ssroeod ii tt pmmoiriggohuhstt  bbaeen dmm oonrraeen ssouu‐siitttraaubbcllteeu rffeoodrr  aaCnnutti‐ibibnaacccottreeprroiiaarall taaepdp pplTliiiccOaat2t iioocnon acctoionmmgspp aabrryee ddM ttooA AOA gg.i. nZZ hahu u neeott vaaelll..   [[6633]] 
pprreeppaaCrreuedd‐c ponoptroaoriunoisunasgn  daelnnedcat nroonl-aysnttero u‐ccsotturnurteacidtnuiCnreugd -i 0n.Cc0o5ur ‐piMnor caoβtr‐epGdoPTr, aiOt0e.21dc  oMTat iiOnC2gA s cboaynatMdi nA0g.Os0 5i bnMya  nMcoovApepOleC r uina-cc oeatna ttaeni noivnegl  
Celue‐cctor(oCnltuya(tiCenHinc3oCgn OteOali)en2c)i tnartgo 1ly60.t.5e0  A5c∙dMomnt−β2a if-noGrin P4,g m 0i.0n1..0 TM5h eMC reA suβal‐tGn sdPho, 0w0.e0.d15  tMhMat  cCCoupA‐Tp ieOarn2 acdoc aet0tia.n0tge5s  d(MCisup (lcaCoyHpedp CerorO uOgahc) et)ataet 
3 2
micropores or crater structures with diameters of 3–5 μm (Figure 10a), and the coatings were fully 
(1C6.u5(CAcHo·vd3eCmreO−dO2 w)f2oi)tr ha 4tn 1ma6n.io5n‐ .gArTa∙dihnmes −rw2e fisotuhrl  t4a  smshizoinew . oeTfdh 3et0 h–rae5s0tu Cnltum s- hT(ioFOiwg2uercdeo  ta1ht0aibnt) g Casut d‐aTi sihpOilg2a hyceoerad mtirnaoggunsg idfhiicsamptilioacnry.o epTdho rero eusgohr 
mcriactreorpsotrruesc tourr ecsrawteirt hstdruiacmtuerteesr swoitfh3 d–5iaµmmete(Frsig oufr 3e–150 μa)m, a (nFdigtuhree c1o0aat)i,n agnsdw tehree cfoualtlyincgos vwereerde fwuiltlyh 
antimicrobial activity of Cu‐TiO2 coatings was improved due to the inhibitory effect of Cu. 
cnoavneor-egdra winisthw inthanaos‐igzreaionfs3 0w–i5t0h nam s(iFzieg uorfe 3100–b5)0a tnamh i(gFhigeurrme a1g0nbifi) caatt iao nh.iTghheera nmtiamgincirfoicbaiatiloanc.t iTvihtey 
Meanwhile, the osteoblastic adhesion, spreading, early proliferation and late differentiation on  
antimicrobial  activity  of  Cu‐TiO2  coatings  was  improved  due  to  the  inhibitory  effect  of  Cu. 

Meanwhile, the osteoblastic adhesion, spreading, early proliferation and late differentiation on