Modificarea suprafetelor implantabile, in vederea cresterii bioperformantelor.

Anodizarea electrochimica , mijloc de modificarea suprafetetelor metalice implantabile

de la micro la nanostructuri.I. Demetrescu

Curs POSDRU

University Politehnica Bucharest, University Politehnica Bucharest, ROMANIA ROMANIA Faculty of Applied Chemistry and Materials ScienceFaculty of Applied Chemistry and Materials Science

Motivatie si suport

• Motivatie : necesitatea de a imbunatati biomaterialele implantabile in contextul dezvoltarii acoperirilor de suprafata

• Suport : Proiecte si colaborari • C.N.C.S.I.S.TIPA. „Obtinerea si caracterizarea de noi micro si nanostructuri compozite cu utilizare in

ingineria tisulara”• Elaborarea si testarea in vitro si in vivo a unor elemente de protezare pentru ortopedie, realizate din

noi biomateriale romanesti • Bilaterala Franta Brincusi «Couches minces d'oxyde d'aluminium et d'oxyde de titane pour

différentes applications technologiques et biomédicales• Proiect CEEX Micro si nanostructuri obtinute prin bioactivare chimica si electrochimica cu aplicatii in

medicina regenerativa• Proiect PN2 IDEI Studii exploratorii asupra mecanismului de formare si inducere de noi proprietati

unor electrozi modificati cu forme structurale TiO2 nanotuburi / nanoparticule si compozite polimerice

• Proiect PN2 IDEI complexe PCCE Noi concepte si strategii pentru dezvoltarea cunoasterii unor

noi structuri biocompatibile in bioinginerie

BiocompatibilitateBiocompatibilitate

Biodegradare

Sterilizare si operatie

Reactia chimicaLa implant

Reactia biologicaLa implant

Toxicitate

Design

Sistemul de parametri” de care depinde biocompatibilitatea

prima interfata intre biomaterial / tesut

cel mai important factor pentru succesul implantului

Conditii dinamice din mediul biologic ⇒ schimbare continua a biosuprafetei - la fiecare pas, materialul este condiţionat de componentele fluidelor biologice.

BiosuprafaBiosuprafaţţa determinanta in a determinanta in evaluarea bioperformanteievaluarea bioperformantei

Aria Aria - determină spaţiul disponibil pentru celulele care vor adera

integrare tesut arie cat mai mare crestintegrare tesut arie cat mai mare cresterea porozitatiierea porozitatii(in limita in care rezistenta materialului permite)

Balanta hidrofil/hidrofob Balanta hidrofil/hidrofob

-- suprafata hidrofilasuprafata hidrofila –– este un merit sau nu ?este un merit sau nu ?;

-- încurajeaza formarea unui film stabil fluid pe material care va descuraja aderarea bulelor de gaz şi a bacteriilor, dăunătoare vieţii implantului

-- unghi de contact mic --suprafata hidofobasuprafata hidofoba -- face ca picătura de apă să ude suprafaţa pe o porţiune cât mai

mică, deci unghiul de contact să fie maxim

CristalinitateaCristalinitatea - poate determina un răspuns specific la proprietăţile mecanice si termice; poate stimula activitatea celulelor

Parametrii biosuprafeteiParametrii biosuprafetei

Metode de modificarea suprafeţei• Stratul modificat obţinut

• Metode mecanice îndepărtează oxizii si murdaria.• - polisare- şlefuire- distrugere suprafaţă lucioasă sau rugoasă

• Metode chimice îmbunătăţeşc biocompatibilitatea, bioactivitatea si conductivitatea osului

• tratament acid strat de oxid cu grosime < de 10 nm• tratament alcalin trat de gel de titanat de sodiu de ~ 1 μm• tratament cu H2O2 strat intern dens şi unul poros extern de 5 nm

• acoperiri sol-gel film subţire de ~ 10 μm cum ar fi fosfatul Ca, TiO2• depunere chimică din fazǎ de vapori (CVD) film subţire de ~ 1 μm de TiN, TiC, TiCN, C

Modificare biochimicămodificari prin silanizarea titanului, fotochimie, depunere de monostraturi, de proteine rezistente, asigura compatibilitatea cu sângele si induce răspunsuri specifice celulelor şi ţesuturilor prin imobilizarea la suprafaţa proteinelor, peptidelor sau factorilor de creştere

Metode fizice •pulverizare termică•pulverizare în flacără•pulverizare în plasmă •ablaţie laser•depunere fizică din fazǎ de vapori (PVD)•Evaporare placare cu ioni împrǎştiere•implantare şi depunere de ioni

• Metode electrochimice• 1. oxidare anodică• 2 depunerea de filme

MOCVD deposition of TiO2 on Ti ( 1) and TiAlNb (2)

• precursor was [TiOCH(CH3)24] titanium isopropyl oxide

• The TiO2 MOCVD surface on Ti which is rutil and anatase (S. Popescu, I. Demetrescu, V.Mitran, A.Gleizes, Molecular Crystal & Liquid Crystal., 483, 266-274, 2008 ;

S. Popescu, I. Demetrescu, C. Sarantopoulos, A.Gleizes, D. Iordachescu, J Materials Science:Materials in Medicine, 18,10, 2075-2083, (2007)

• The α+ β phase coresponding to ternary TiAlNb is not visible, the image being more close to SEM image of Ti coverd with a TiO2 film.

• The emission X-ray spectrum presenting elemental composition confirms presence only for Ti and O

SEM micrographs of modified TiAlNB surface via MOCVD ;

a) TiAlNb/TiO2 – T 500/p 20/χ 76 ;b) TiAlNb/TiO2 – T 400/p 1/χ 5000

Laser ablation

• The SEM image of ablated Ti6Al7Nb surface indicates formation of a continuous oxide covering the entire surface and having a granular structure with diameter from 1 μm to 3 μm.

• the surface is not an homogenous and compact one.

• Between bigger grains which are conglomerates of smaller grain with a diameter around 0.6µm, appear vacancies as cracks;according to EDAX spectrum the formed oxides are TiO2, Al2O3, Nb2O5

TiO2 ca micro si nanostructuri pentru aplicatii medicaleTiO2 ca micro si nanostructuri pentru aplicatii medicale

Micro and nano acoperiri

Inorganic filmsInorganic films Organic filmsOrganic films

Filme pasive asive TiO2

• TiO2 nanotuburi bbiopolymeric films

Depinzand de geometria nanotuburilor si de caracDepinzand de geometria nanotuburilor si de caracteristicile de teristicile de suprafata suprafata •• Nanotuburi pentru aplicatii bio (crestere si adeziune celularaNanotuburi pentru aplicatii bio (crestere si adeziune celulara

• Nanotuburi pentru fotocataliza, nanotuburi pentru autocuratire , etc

Filme bioactive (calcium phosphates)

Hybrid films( organic +inorganic )

Filme cu polimeri conductori si nanoparticule

Resistente la corroziune si prietenose pentru organismele vii

Ti si aliaje Ti

modificari la suprafata

Natural passivation of Ti and Ti alloy

A mixture of oxides, predominant TiO2

TiO, T2O3, TiO2 etc • Nonstoichiometric, • amorphous, • Insoluble

• titanium oxide layer (3-7 nm thick) forms spontaneously on the Ti or Ti-alloy surface upon contact with air.

Ringer 1, 8.6g/L NaCl; 0.33g/L CaCl2; 3g/L KCl

Ringer 2, NaCl 0.3 g/L, KCl 0.37 g/L, NaHCO3 2.44 g/L, MgCl 2.6H 20.203 g/L, MgSO4.7H2O 0.123 g/L,Na2HPO412H2O 0.07 g/L and NaH2PO4.H 20.069 g/L

SBF (142 mMNa+, 5mM K+, 2.5 mM Ca 2+, 1.5 mM Mg 2+, 147.8 mM Cl-, 4.2 mM HPO4 2-, 0.5 mM SO42- ).Hank, NaCl 8 g/l ; CaCl2 0.14 g/L; KCl 0.4 g/L; MgCl2.6H20 0.1g/L Na2HPO4. 12H2O 0.06 g/L ;MgSO4.7H2O 0.06g/lL glucose 1g/L

NaCl 0,9 %Artificial saliva Tani Zuchi, KCl 1.5g/L; NaHCO3 1.5 g/L; NaH2PO4 0.5g/L; KSCN 0.5g/L; lactic acid 0.9g/L.Artificial saliva Afnor NaCl O.7g/L, KL 1.2 g/L ,Na2HPO4, 0.26g/L ,NaHCO3 1.5g/L,uree 1.3g/LLactic acid, Albumin, The temperature were; -generaly 37°C, the temperature of the human body

-for dental media various temperatures ( from ice cream to hot soup )

BIOLOGICAL ENVIRONMENTS

4504003503002500 50 100 150 200-800

-600

-400

-200

0

200

TiTi6Al4VTi5Al2,5Fe

E(m

V)v

s A

g/A

gCl

time (days)

Variation of potential in time in Ringer 2 Variation of potential in time in Ringer 2 (Natural passivation )

Variation of ion release in time in Ringer 2 solution

10 15 20 25 300

1

2

3

4 Ti Ti6Al4V Ti5Al2,5Fe

ion

rele

ase

(ug/

cm2 )

time (h1/2)

Y = 0.53963+0.11561x

Y=-0.9081 + 0.14469x

y =1.3965+0.0271x

Y =-0.4986+0.0382x

Correlation between surface treatment, local susceptibility to Corrosion and Roughness in Ringer 2 solution:

first sample, larger roughness values (281.4006nm) and important difference

EBr - EPr (1000mV) is observed; second sample, no breakdown was registered and the average roughnes has lower values (194.71 nm).

AFM for Ti-5Al-2.5Fe electrodes alloys, chemically

polished in 3% HF+20%HNO3 (immersion 20 days in Ringer 2

AFM for Ti-5Al-2.5Fe electrodes alloys treated with ultrasonic procedure (immersion 20 days in Ringer 2)

Bioethics and animal experiments in biomaterial evaluation

• The 3 R• Refinement of procedures in order to minimise animal

suffering and distress.• Reduction of the numbers of experiments • and number of animals used• Replacement of animal experiments with • non-animal alternatives • Because of the 3R problems of animal experiments, in vivo

experiments tend to be more and more limited

• In vitro experiments become more important

Biocompatibility (cytotoxicity test )

• cytotoxicity test using direct contact method, with secondary cultures of human skin fibroblasts (HSF), obtained, grown, and subculturedat 37oC in a humidified incubator equilibrated with 5% CO2.

• The cells were seeded on the films at a density of 1 ×105 cells/ml and cultured for up to 7 days. Fibroblast proliferation was assessed by measuring the mitochondrial dehydrogenase activity.

MTT AssayMTT AssayMTT AssayMTT Assay measures the cell activity, proliferation rate and cell measures the cell activity, proliferation rate and cell

viability. The yellow tetrazolium MTT (3viability. The yellow tetrazolium MTT (3--(4, 5(4, 5--dimethylthiazolyldimethylthiazolyl--2)2)--2, 52, 5--diphenyltetrazolium bromide) is reduced by metabolically active diphenyltetrazolium bromide) is reduced by metabolically active cells, cells, in part by the action of dehydrogenases, to the corresponding blin part by the action of dehydrogenases, to the corresponding blue ue formazan. formazan.

The formazan crystals were solubilized with DMSO and absorbance The formazan crystals were solubilized with DMSO and absorbance of of the supernatant was measured at 550 nm. Absorbance values that athe supernatant was measured at 550 nm. Absorbance values that are re lower than the control cells indicate a reduction in the cell aclower than the control cells indicate a reduction in the cell activity and tivity and viability. Conversely, a higher absorbance rate indicates an incviability. Conversely, a higher absorbance rate indicates an increase in rease in cell activity/proliferation.cell activity/proliferation.

Cell viability

0

2 0

4 0

6 0

8 0

1 00

1control Ti Ti CVD 34 Ti CVD 7 Ti colagen

Viability of cells depends of the treatment of substrate

Biocompatibilitate (HGF cells)

The imunofluorescent signals revealed a good fibronectine expression. Usually fibroblasts prefer smooth surfaces but recent papers sustained that microroughness on cellular response is inconclusive taking

into account the etching.

Cell alignement and cytoskeleton organization by actin labeling with phalloidinI.

Demetrescu, C. Pirvu,and V. Mitran, Effect of micro and nano - topographical features of Ti/TiO2 electrode surface on cell response, Bioelectrochemistry 79, 122-129, (2010)

Histogram for distribution of cell’s size

Majority of cells has long axis size between 5-10 μm and there are a few with diameter between 15-20 μm or 20-30 μm(probably they represent groups of two or three cells.

Total image area = 305410,56 μm2

Total area occupied with cell = 46542,4 μm2

Cell spreading: 15,23 %

20 μm

Image Analysis in cell culture research

Analysis was performed with the Sigma Scan program to estimate cell spreading.

Oxidarea anodică• procesul prin care, în prezenţa unui câmp electric, la anod au loc reacţii prin

care ionii de oxigen difuzează la metal şi formează un film de oxid pe suprafaţa anodului.

• este cea mai stabilă metodă folosită la obţinerea diferitelor tipuri de straturi de oxizi protectori pe metale.

• anodizarea Ti este folositǎ pentru creşterea grosimii stratului de oxid;• în scopul îmbunătăţirii protecţiei împotriva coroziunii şi micşorării cantităţi de

ioni metalici eliberaţi, • pentru obţinere de acoperiri poroase in vederea îmbunătăţirii adeziunii şi legării

de os în aplicaţiile biologice.

• proprietăţile structurale şi chimice ale oxizilor anodici pot fi schimbate prin variaţia parametrilor de proces, cum ar fi: potenţialul anodic, compoziţia electrolitului, curentul si temperatura.

Cresterea filmului de oxid• Parametrii electrochimici influenţeazǎ critic proprietǎţile şi creşterea în grosime a

oxidului pe suprafaţa de titan. • aceastǎ creştere modificǎ topografia suprafeţei, în special configuraţia suprafeţei

poroase iar un anion încorporat în stratul de oxid altereazǎ compoziţia chimicǎ şi structura cristalinǎ a oxidului de titan, reprezentatǎ de anatas şi rutil ;

• rezultatele raportate în literaturǎ prezintǎ date contradictorii asupra asupra comportamentului creşterii electrochimice a filmului anodic de oxid.

• Ti se comportǎ ca un metal supapǎ, creşterea de oxid implicând migrarea ionilor prin filmul de oxid,

• creşterea grosimii stratului de oxid este guvernată de legea lui Faraday.

• Filmul de oxid obţinut trebuie să fie uniform ;acest aspect influenţeazǎ considerabil proprietǎţile filmelor : profilul defectelor şi densitatea lor precum şi formarea unui film de suboxid intermediar TiO2-x între filmul TiO de pe suprafaţa substratului de titan şi filmul superficial de TiO2 al stratului de oxid

Reacţiile principale la oxidarea anodica• În timpul oxidǎrii anodice a titanului se observǎ o evoluţie a gazului (O2 şi H2), care

reduce eficienţa creşterii stratului de oxid ; structura filmului anodic se modificǎ din fazǎ amorfǎ în fazǎ cristalinǎ, ex. anatas şi rutil.

• Aceastǎ transformare cristalograficǎ este relatatǎ de fenomenul „breakdown”, care este dependent de parametrii electrochimici, cum ar fi concentraţia de electrolit (sau activitatea) şi densitatea de curent.

• Reacţiile principale care au loc la oxidarea anodică sunt următoarele: • La interfaţa Ti / oxid de Ti:• Ti ↔ Ti2+ + 2e-

• La interfaţa oxid de Ti / electrolit:• 2H2O ↔ 2O2- + 4H+ (ionii de oxigen reacţionează cu Ti şi formează oxid)• 2H2O ↔ O2 (gaz) + 4H+ + 4e- (oxigenul gazos se degajă sau se absoarbe pe

suprafaţa electrodului) • La ambele interfeţe are loc urmǎtoarea reacţie:

• Ti 2+ + 2O2-↔ TiO2 + 2e-

Oxidul de titan• Oxidul de titan format anodic are rezistivitate relativ ridicată faţă de electrolit şi părţile

metalice ale circuitului electric.

• Grosimea finală a stratului de oxid, d, variază aproape liniar cu tensiunea aplicată, U, conform relaţiei d = a·U, (a este o constantă cu valori cuprinse între 1.5 ÷ 3 nm V-1.

• Dacă anodizarea se face la o tensiune mai mare faţă de limita de „breakdown”, oxidul obţinut nu este suficient de rezistent pentru a preveni scurgerea ulterioară de curent. Acest tip de anodizare se referă adesea la anodizare în puls, care duce la formarea unor filme poroase de oxizi.

• Cuia obţine TiO2 anodic, utilizând ca electroliţi H2SO4 H3PO4 şi Na2SO4 prin impunerea unor tensiuni diferite timp de 1 minut, la temperatura camerei. În urma anodizǎrii, filmele obţinute sunt alcătuite din faze de rutil şi / sau anatas cu structurǎ poroasǎ în cazul electroliţilor H2SO4 şi Na2SO4, în timp ce, în electroliţii CH3COOH şi H3PO4 filmul de oxid de titan este amorf.

• S-a observat cǎ titanul acoperit cu filme tip rutil şi / sau anatas stimuleazǎ formarea unui strat compact de apatitǎ dupǎ imersarea acestuia în SBF timp de 7 zile, în timp ce, titanul acoperit cu un strat amorf de oxid de titan nu induce formarea apatitei

Bioliquid ECor(mV)

I Cor(μA/cm2)

I pas(μA/cm2)

E br(mV)

E pr.(mV)

E br –E pr(mV)

A. Ringer1 -250 1,3 12

A. Ringer2 -180 0,01 4,8

A. Hank -165 0,011 26

A. NaCl 0,9 % -180 3,307 13,6 2946 2356 590

B. Ringer1 -537 1.1 11

B. Ringer2 -519 0,02 0.62

B. Hank -475 0,31 0.68

B NaCl 0,9 % -545 0,8 18 1325 1050 375

Electrochemical parameters from polarization curves of Ti6Al4V Electrochemical parameters from polarization curves of Ti6Al4V samples (anodized )samples (anodized ) ;;A data before surgical intervention A data before surgical intervention

B data 6 months after surgical interventionB data 6 months after surgical intervention

• Dupa pregatirea suprafetei ,pentru anodizare se foloseşte o celulă electrochimică cu doi electrozi; anodul este Ti6Al7Nb iar catodul Pt. Tensiunea anodică este de 20 V timp de 1 oră. Variaţia curentului la anodizare din soluţii H2SO4 1 M, H3PO4 1 M şi HF 0.25 M pe un electrod de Ti6Al7Nb, este :

•

0 1000 2000 3000 4000

0

200

400

600

i,mA/

cm2

Timp, minute0 1000 2000 3000 4000

0

200

400

600

i,μA

/cm

2

Timp, minute 0 1000 2000 3000 4000

0

200

400

600

i,μA/

cm2

Timp, minute

Cronoamperogramele în H2SO4 1 M şi H3PO4 1 M pot fi divizate în două domenii. La început, densitatea de curent scade exponenţial odată cu formarea unui strat de oxid compact după care devine constantă ca urmare a creşterii stratului de oxid pe aliaj.

In HF existâ trei faze: prima este caracterizatǎ de o scǎdere exponenţială a curentului, faza a II-a de creştere a curentului şi apoi stabilizarea acestuia – faza III. Anodizarea Ti este însoţitǎ de dizolvarea chimică a oxidului de titan datorită formării TiF6

2-.

strat 280 nm cu pori mari peste care se formeaza strat fosfat

Obţinerea structurilor oxidice din trei soluţii de electrolit: H2SO4 1 M, H3PO4 1 M şi HF 0.25 M

Parametrii de coroziune obţinuţi din diagramele Tafel

Parametrii Ti6Al7Nb netratat, anodizat în H2SO4 anodizat în H3PO4 anodizat în HF

Icor (A/cm2) 8.12·10-8 3.662·10-9 2.485·10-9 5.584·10-9

Ecor (mV) 0.422 -0.161 -0.199 -0.115

Rp (Ω·cm2) 3.25·10+5 5.771·10+6 7.586·10+6 1.959·10+6

vcor(mm/an) 6.462·10-4 3.137·10-5 2.129·10-5 4.783·10-5

EISCircuitul echivalent pentru aliajul Ti6Al7Nb

netratat, este alcătuit prin combinarea în paralel a unui rezistor, corespunzător rezistenţei de transfer de sarcină (RbL) şi din elementul de fază constantă, corespunzător filmului de oxid barieră (CbL).

• Elementele de circuit RpL şi CpL, reprezintă rezistenţa stratului de oxid obţinut în urma tratamentelor aplicate şi respectiv, elementul de fază constantă corespunzător filmului de oxid obţinut. Rs este rezistenţa soluţiei Hank.

• Rezultatele procesului de fitare susţin procesul de difuzie prin stratul de oxid obţinut în urma anodizării.

circuitul echivalent folosit la fitarea spectrelor obţinute pentru Ti6Al7Nb anodizat din soluţiile de electrolit: H2SO4 1 M, H3PO4 1 M şi HF 0.25 M.

a TiAlNb netratat B anodizat în

H2SO4 1M

C anodizat în H3PO4 1M D anodizat

în HF 0.25M

Ra (rugozitatea medie) pentru a= 16.39, pentru b= 20.6, pentru c=14.5 si pentru d =78.5 : unghiurile de contact sunt respectiv 85.89, 50.68, 30.34 si 63.08

Stratul de oxid obţinut anodic pe Ti• Stratul de oxid obţinut anodic pe aliajul de titan poate fi sintetizat fie prin

folosirea acidului fosforic, fie a electroliţilor apoşi care conţin calciu şi fosfor dizolvaţi

• rezistenţa la coroziune creste cu timpul de oxidare, dar apar defecte pe suprafaţǎ.

• Pentru intensificarea inducţiei osoase şi a conducţiei în jurul implantului se depune TiO2 prin imersarea titanului în soluţie apoasă de NaOH şi încălzire la 400 °C

• Filmul de oxid de titan (titania) induce formarea osului prin obţinere de apatită pe suprafaţa titanului când acesta este imersat în fluid fiziologic simulat (SBF), care conţine aceiaşi ioni anorganici şi are concentraţia asemǎnǎtoare plasmei sângelui uman. Prin tratarea titanului cu TaCl5 cu conţinut de peroxid de hidrogen (30 %; H2O2yTaCl5) se asigurǎbioactivitatea in vitro

TiO2 Nanotubes fabrication • Nanobiomaterialele au un numar crescut de atomi la suprafata si poseda un raport mai mare

suprafata la volum fata de materialele conventionale.

Generally, there are three strategies used in the fabrication of TiO2 nanotubes:

I. Template synthesis II. Hydrothermal methods, andIII. Electrochemical synthesis (anodizing of pure Ti foils)

• Anodizing was a choice for a chip and convenient way[1-3].

• Changing anodizing parameters as voltage, pH electrolyte compositions and time of electrolysis a large variety of structure with various potential applications could be identify.

. 1.S.P. Albu, A. Ghicov, J.M. Macak, P. Schumuki, Phys. Status Solidi 1, (2007) R–65-R-67. .2. A. Ghicov, H. Tsuchiya, R, Hahn, J.M. Macak, A. G. Munoz, Electrochem. Commun., 2006, 8, 528.3. Man, C. Pirvu, I. Demetrescu, Rev. de Chimie, 59(6 ), (2008), p 615.

METHODOLOGY

Methods in fabrication and in characterization of TiO2 nano tubes • Fabrication: Anodizing, • Characterization• Electrochemical stability

open circuit determinations, cyclic voltametry, in bioliquids as NaCl,Hank and SBF(simulated body fluids )

• Surface analysis methodsScanning electronic microscopy (SEM), Atomic force microscopy ( AFM) and Contact angle determination (CA)

• ICP-MS (inductively plasma mass spectrometer) measurements for ions release determination

• Biocompatibility tests

TiO2 nanotubes electrochemical synthesis

• The TiO2 nanotubes electrochemical synthesis can be divided in four generations as following :

• 1) first nanotubes generation elaborated in HF based electrolytes;• 2) second nanotubes generation fabricated in buffered electrolytes; • 3) third generation of nanotubes obtained in polar organic electrolytes;• 4)fourth generation based on anodizing in electrolytes without fluorideSamples preparation • Titanium foils samples (2 mm thickness) were mechanically polished, degreased in

acetone, washed for five minutes in de-ionized water and then immersed in a HF and HNO3 solution for 30 seconds. Then washed again with de-ionized water for five minutes and dried at 40° C.

Ti Anodizing mechanisms

TiO2

Ti

Anodizing mechanism in usually used electrolytes consists in a oxide passive stratum formation

Mechanism of nanotubes formation;from passivity breakdown to nanopores and nanotubes ; in fluoride mixtures, an oxide layer is formed on the surface of titanium as well:

Ti +2H2O→ TiO2 +4H+However, in the presence of F- the oxide layer partially dissolves and forms pits according to the reaction TiO2 +6HF→ [TiF6]2- +2H2O + 2H+.These concurrent processes anodic oxidation and dissolution leads to the formation of nanotube.

Nanotubes stages formation: from compact oxide to autoorganized layers.

Anodizing conditions used for obtaining TiO2 nanotubes

Anodizing conditions Sample Electrolyte V t, min T, oC

S1 5 30 S2 10 30 S3 15 30 S4 20 30 S5 5 50 S6

1:7 volumetric ratio CH3COOH + 0.5 %

HF 10 50

Room

temperature

S3

S1

S6

Stage 1: pores nucleation at TiO2

compact oxide

S4

Stage 3: widening and deepening of the pores in the initial porous stratum

S3

Stage 4: transition from pores to nanotubes is almost complete

S1

Stage 5: auto-

organized nanotubes

layers

S6

Stage 2: initial Poresgrowth

S4

AFM structure of the a) native passive film b) nanotubes selforganized layer

a) b)

The Root Mean Square (RMS) parameter and Roughness Average, Rawere 66.8 nm and 82.7 nm for a). For b) the values are 23 and 29 nm

SEM of TiO2 native passivatedTiO2 nanotubes selforganized layer (110 nm nm diameter

AFM images of nanotubes on TiAlNb

Three - dimensional AFM images for Ti6Al7Nb alloy [1](a) untreated; (b) covered with oxide layer obtained in (NH4)2SO4 1 M + NH4F 0.5 wt%

solution and with roughness as Ra=83 nm and RMS=104,3nm(c) covered with oxide layer obtained in glycerol + 4% H2O + NH4F 0.36 wt%

mixture and with roughness as Ra=68 nm and RMS=90nm[1]M. Mindroiu, C. Pirvu R. Ion I. Demetrescu Comparing performance of nanoarchitecture fabricated by Ti6Al7Nb anodizing in two kinds of electrolytes,

Electrochimica Acta vol 56,1, 15 December 2010, Pages 193-202

ab c

Anodizing conditions• The anodizing procedure was performed at room temperature for 2 hours;

- at various constant voltage;- in different electrolytes with fluoride ions

• Electrolyte Voltage Time Nanotubes CAComposition

diameter (nm)1.HF 0.5 % 20 V 120 minutes 80-160 83.392. HF 1% 20 V 120 minutes 1003.0.5% HF + 5 g/l Na2HPO4 20V 120 minutes 60-75 55.454.HF 0.5%+5g/l NH4F 20V 120 minutes 605.HF 0.5% + H3PO4 1M 20V 120 minutes 400 77.45 6.(NH4)2SO4 + 0.5 wt.% NaF 20 V 120 minutes 120 16

7. glycerol +0.36M NH4F 20 V 120 minutes 40-55 49.97 8.glycerol +0.36M NH4F 40V 120 minutes 65-75

SEM measurementsWith the increase in voltage, the ordered structures shift from nanotubular aspect towards a

porous distribution. At 20V applied voltage the nanotubes have a clear an ordered aspect. At 40V, the nanotubes are incorporated by porous structures of the same diameter. The incorporation process accentuates for the higher voltage and at 80 V applied voltage gives a final porous-only surface.a) b)

c)

Surface characteristics as a function of anodizing

electrolyte composition

No. Electrolyte composition Ra (nm) Diameter (nm) Contact angle

1 HF 0.5% 0.9 100 83,39

2 HF 0.5%+5g/l NH4 0.36 60 55,45

3 HF 0.5%+H3PO41M 0.2 400 77,45

4 (NH4)2SO4+0.5 NaF% 23 120 16

5 1:7 volumetric ratioCH3COOH + 0,5% HF

17,6 20-30 73,7

6 16,4 18-23 50,6

A top nanotubes are bundling together as a consequence of chemical etching

One step anodization

One step at 15 V for 3 h

the nanotubes grown at 30 V for 1 h and then at 15 V for 6 h the tube length of the bottom layer increases with anodizing duration

aunprotected single layer grown at 15 V for 6 h in the electrolyte used once at 30 V for 1 h

A Two-step anodization to grow high-aspect-ratio TiO2 nanotubes(Xiaoyan Wang, Sam Zhang , Lidong SunThin Solid Films xxx (2011) xxx–xxx

A two-step anodization by switching voltage from high to low effectively grow high-aspect-ratio TiO2 nanotube arrays. As highvoltage is applied first and low voltage second, large diameter tubes form at the top and small diameter tubes form underneath. In this structure, the top layer serves as a sacrificial layer and undergo chemical dissolution which effectively protects the growth of the underneath tubes of small diameter. As low voltage is applied first and high voltage second, tubes of small diameter form on the top and largediameter tubes form underneath. In this manner, the top layer suffers fast chemical dissolution thus cannot be used as an effective protector.

The surface morphology of TiO2 nanotubes grown at 15 V

• The tubes are uniform with an average inner diameter of 35±3 nm and outer diameter of 45±4 nm. The value is close to the estimated ideal tube diameter of 30 nm which could provide comparable specific surface area with nanoparticles [1].

• Frank and all [2] reported that nanotubes with an average inner

diameter of 30±4 nm have comparable surface area and dye loading with nanoparticle films with a particle size of ~24 nm for a given film

thickness.

1.L. Sun, S. Zhang, X.W. Sun, X. He, in: S. Zhang (Ed.), Handbook of NanostructureThin Films and Coatings, vol. 3, CRC Press, New York, 2010, p. 57.

2. K. Zhu, N.R. Neale, A. Miedaner, A.J. Frank, Nano Lett. 7 (2007) 69.

In-situ preparation of multi-layer TiO2 nanotube array thin films by anodicoxidation method (Materials Letters 65 (2011) 1188–1190 )

Cross section of multi-layer TiO2 nanotube array thin films fabricated under anodic voltage: shifting from 30 V to 10 V (a) and from 60 V to 10 V (c), cross section of TiO2 nanotube array thin films formed at the anodic voltage of 30 V (b) and 60 V (d).

TiO2 nanotubes performances evaluation (nanotubes 120 nm diameter )

• Results in open circuit • The variation of potential in time in NaCl 0.9%

• Immediately after immersion in NaCl the corrosion potential presents electronegative values, but a tendency to more noble values are taking place for both kind of sample. In both cases after various period of time the potential seems to reach a steady state value, which is in the range of more electropositive domains. for the self organized nanotubes structures.

0 2 4 6 8 10 12 14 16-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

E (m

V) v

s S

CE

time (days)

OCP for samples with a native passive stratum OCP for samples with self organized nanotubes The initial event during the

immersion of Ti in bioliquids, is the hydrolysis of the TiO2 and the establishing of the equilibrium surface-solution. The dissolution products released are neutral species like Ti(OH)2or TiO(OH)2hydroxocomplex.

Sarcina la suprafata depinde de ph-ul electrolitului. La ph mic pH (<4), formarea [TiOH2]+ din grupe TiOH provoaca

incarcare pozitiva;la pH (>9) TiOH conduce la cedare de proton si [TiOH]- (sarcina

negativa). Intre pH 4 si 9, coexista ambele tipuri de reactii la suprafata.

Punctul izoelectric la care sarcina neta pe Ti este 0 (IEP) este 5-6.pentru oxidul de Ti si deci la pH neutru (pH 7), suprafata este usor negativa , reactia de deprotonare fiind preponderenta.

Sarcina la suprafata datorata reactiei TiO2/H2O afecteaza adsorbtia de proteine si alte macromolecule cu rol in adeziunea si proliferarea celulara.Adsorptia de calciu si de ioni fosfat pe stratul de oxid creaza centre de nucleiere pentru formarea si dezvoltarea tesutului osos la interfata implant tesut (carbonate HA ))

Sarcina (-) atrage Ca 2+, Na+, Mg 2+,

Sarcina (+) atrage H2PO4 - or HPO4

2-

Corrosion parameters for nanotubular samples in NaCl 0.9% solution

Nanotubes S1 have diameter around 100nm and nanotubes S2 have diameter around 60nm

Sample icorr ( μAcm-2) Ecorr (mV vs SCE) Ipass (μAcm-2)

Nanotubular S1 185 -1280 245

Nanotubular S2 12 -710 84.5

Remark: smaller corrosion rate for nanotubes with smaller diameter ( size dependent behavior)

Cyclic voltametry for TiO2 nanotubes in Hank solution

Electrochemical parameters from Tafel plots ( a size selective stability, the less stable being the structure with largest diameter as 400 nm)Anodizing Ecor (mV) Icor (μA/cm2 Vcor (μm/y) diameter(nm)conditions vs SCE) 1. HF 0.5% -89,62. 2.31 6.49 80-2.HF5%+5g/l NH4F -62,4 1.97 2.98 60 3.0.5%HF + H3PO4 1M -162,4 9.98 8.85 400.

Corrosion current densities decrease in the following direction:I corr HF0.5% + H3PO4 1M >I corr HF 0.5% >I corr HF 0.5%+5g/l NH4F

The most stable nanostructure is fabricated in HF5%+5g/l NH4F

Nanotubes calcinationnanotubes elaborated in 1:7 volumetric ratio

CH3COOH + 0,5% HFSurface analysis of the obtained samples before and after annealing

Before annealing After annealing

Nanotubes dim. Nanotubes dim. Sample d, nm twall, nm

CA Value d, nm gper, nm

CA Value

Ti ― ― 76.03 ― ― ― S1 20-30 6-11 73.7 10-20 12-18 27.61 S2 25-40 7-12 73.15 18-30 9-17 17.61

Modifications of nanotubes dimensions after annealing

Before annealing After annealing Difference Sample d , nm twall, nm d , nm twall, nm d , % twall, %

S1 21.73 8.06 17.68 14.05 - 18.64 + 74.32 S2 34.10 9.51 22.36 12.9 - 34.43 + 35.65

Corrosion parameters for S1 annealed, S2 annealed and TiO2 nanotubes – nontreated sample and for Ti [17]

Sample Ecor,

mV icor,

μA/cm2 ipas,

μA/cm2 vcor, mm/Y

·10-2 Rp,

kOhmcm2

cp-Ti -606 2.32 8.5 5.7 TiO2

nanotubes -497.3 1.0160 0.0401 1.963 0.574

S1 annealed 666.2 0.6714 0.682 1.297 8.05 S2 annealed 429.5 1.8403 0.579 3.555 18.11

The annealing treatment was done at 5000C for 2 h.

Biocompatibility evaluation• From ability to built phosphate on the surface as a

biomimetic coating• From cell culture

Samples Ecor(mV)vsSCE

Icor (µA/cm2) Vcor (µm/y)

Ti -205 9.6 68,28

Ti anodized in HF0.5%+5g/l NH4F

-62,4 1.97 2.98

Tianodized in HF 0.5%+5g/l NH4Fafter 2 days immersion in SBF

449 0.11 1.2

It is to point that on the native passive TiO2immersed in SBF the same period of time, no phosphate was put in evidence, and that is an argument for ability of this kind of nanostructure to promote surfacebioactivation inducing phosphate formation.

SEM images of film deposited in SBF (142 mMNa+, 5mM K+, 2.5 mM Ca 2+, 1.5 mM Mg 2+, 147.8

mM Cl-, 4.2 mM HPO4 2-, 0.5 mM SO4

2- ).after 2 days on Ti02 nanotubes; the main organic component of bone is a triple helix 300 nm length,0.5 nm in width;HA, the second component has particle sizes around 20-40 nm long and it is possible to assume that bone cells are accustomed to a nanoscale tubular environment rather than the micro-scale environment .

A better ability to form phosphate; nanotube TiO2structure as anchorage of phosphate coating

• a calcium-phosphate deposition was detected with FTIR analysis on the surface electrodes with and without nanotubes. For the coated nanotubes TiO2 in SBF after 2 days immersion (a) and for coated native passive stratum (b) after 3 days immersion;• the presence of carbonate group at around 1430 cm-1, hydroxide group at around 3240 and 1640 cm-1,and phosphate group at 600, 565, 616 cm-1, representing O-P-O bending vibration; the 616 cm-1 band is a suggestion for appearance of a crystalline octocalcium phosphate, a precursor of HA.

50556065707580859095

100

650 1150 1650 2150 2650 3150 3650

Wavenumber, [1/cm]

Tran

smita

nce,

[%]

b

0102030405060708090

100

550 1050 1550 2050 2550 3050 3550

Wavenumber, [1/cm]

Tran

smita

nce,

[%]

a) b)

Sample icorr ( μAcm-2) Ecorr (mV vs SCE) Ipass (μAcm-2)

Nanotubular S1 (diameter 100 nm)

21.5 -885.1 28.82

Nanotubular S2(diameter 60nm

2.03 -598.6 12.4

Corrosion parameters for nanotubular samples in SBF solution

Remark: smaller corrosion rate for nanotubes with smaller diameter ( size dependent behavior)

ICP-MS determinationsIn the time of hydroxyapatite growth on the nanotubes

surfaces the release of Ca2+ and Ti ions was observedAt the first the time of developmentof hydroxyapatite growth on nanotubes surfaces the Ti ionsrelease is smaller comparing tothe rate after 2 days immersion (0625 ppb/ Day ). After 30 days the decrease is more evident the rate being 0.016 ppb/day.

0 5 1 0 1 5 2 0 2 5 3 0

0 .1

0 .2

0 .3

0 .4

0 .5

ion

rele

ase

(ppb

)

t im e (d a y )

T i2 +

C a 2 +

Thank you for your attention