ma e ials
A icle
De elopmen o Biomime ic NiTi Alloy: In luence o
The mo-Chemical T ea men on he Physical,
Mechanical and Biological Beha io
Elisa Rupé ez 1,*, José Ma ía Mane o 1, Luis-Albe o B a o-González 2, Edua do Espina 3
and F.J. Gil 4
1
Cen e de Rece ca Nanoenginye ia (C nE), Depa amen o Ciencia de los Ma e iales e Ingenie ía Me alú gica,
Escola Tècnica Supe io d’Enginye ia Indus ial de Ba celona (ETSEIB),
Uni e sidad Poli écnica de Ca alunya, Ba celona 08028, Spain; jose.ma ia.mane [email p o ec ed]
2Unidad Docen e de O odoncia, Facul ad de Odon ología, Uni e sidad de Mu cia, Mu cia 30003, Spain;
[email p o ec ed]
3Depa men o O odoncia, Facul ad de Odon ología, Uni e sidad de Se illa, Se illa 41009, Spain;
[email p o ec ed]
4Uni e sidad In e nacional de Ca aluña, C/Immaculada 22, Ba celona 08195, Spain; xa ie [email p o ec ed]
*Co espondence: elisa. upe [email p o ec ed]; Tel.: +34-9340-16712
Academic Edi o : E ik Reimhul
Recei ed: 6 Ap il 2016; Accep ed: 17 May 2016; Published: 24 May 2016
Abs ac :
A bioac i e laye , ee o nickel, has been pe o med o i s g ea e accep abili y and
eliabili y in clinical applica ions o NiTi shape memo y alloys. In he i s s ep, a sa e ba ie
agains Ni elease has been p oduced on he su ace by means o a hicke u ile/anas ase p o ec i e
laye ee o nickel. In he second s ep, a sodium alkaline i ana e hyd ogel, which has he abili y
o induce apa i e o ma ion, has been pe o med om oxidized su ace. An imp o emen o hos
issue–implan in eg a ion has been achie ed in e ms o Ni ions elease and he bioac i i y o he
ea ed NiTi alloys has been co obo a ed wi h bo h
in i o
and
in i o
s udies. The ans o ma ion
empe a u es (A
s
, A
, M
s
, and M
), as well as he c i ical s esses (
σβôM
), ha e been sligh ly changed
due o his su ace modi ica ion. Consequen ly, his ac mus be aken in o accoun in o de o design
new su ace modi ica ion on NiTi implan s.
Keywo ds: bioac i i y; NiTi alloy; supe elas ici y; shape memo y e ec ; biomime ic su aces
1. In oduc ion
NiTi shape memo y alloy (NiTi SMA) is an in e es ing ma e ial in he biomedical a ea due o
i s unusual p ope ies such us one-way and wo-way shape memo y e ec s, supe elas ic e ec , high
damping p ope y and ubbe -like e ec [
1
]. Fo hese easons, NiTi SMA has been used o di e en
o hopedic applica ions such as load-bea ings pla es o bone ac u e epai , in e nal ixa ions o
long bone sha s, spinal co ec o s, e eb al space s o bone dis ac ion de ices [
2
,
3
]. Howe e , o
long- e m medical ou comes, some conce ns s ill emain o be add essed; o example, i s po en ial
oxici y and bioac i i y [4–6].
Conce ning oxici y, i is well epo ed ha nickel ions elease om he su ace (Ni dissolu ion o
wea deb ies) may cause some alle gic eac ion and biocompa ibili y p oblems [
4
–
6
]. The p o ec i e
nickel- i anium oxide laye ac s as a ba ie o chemical a ack and co osion and con ines he di usion
o Ni ions, howe e , his oxide laye can be deple ed by wea , co osion and a igue. By means o su ace
oxida ion (lase o plasma sou ce ion implan a ion echniques), i is possible o educe he amoun o
Ni ions leaching om he su ace o NiTi implan o negligible amoun s [
7
,
8
]. Ne e heless, one
o he majo issues has p o en o be an inadequa e in e ac ion be ween he NiTi SMA and he
Ma e ials 2016,9, 402; doi:10.3390/ma9060402 www.mdpi.com/jou nal/ma e ials
Ma e ials 2016,9, 402 2 o 11
su ounding na u al issue leading o o eign body eac ions [
9
]. Fo example, he appea ance o
a dense laye o ib o ic connec i e issue a in e ace o bone–implan could gene a e weak ancho age
be ween he implan and adjacen bone leading o mic o-mo ion and, inally, loosening o he implan .
The e o e, p epa a ion o a bioac i e laye , ee o nickel, is ega ded as a c i ical issue o i s g ea e
accep abili y and eliabili y in clinical applica ions.
De elopmen o calcium phospha e laye s on implan s su ace a e conside ed o be one o he
p ospec i e me hods o imp o ing bioac i i y [
10
–
12
]. Di e en su ace echniques, o example,
he mal sp ay, physical apo deposi ion, elec odeposi ion o sol-gel echniques, ha e been employed
o p o ide me al implan s wi h bone-bonding abili y [
13
–
15
]. Mediaswan i e al. [
16
] p esen ed
a summa y o he cha ac e is ics o he a ious coa ing echniques o calcium phospha e showing i s
own bene i s and d awbacks espec i ely.
Recen ly, i has been epo ed ha he de elopmen o biomime ic su aces by he mochemical
ea men s is a p omising me hod due o i s abili y o p oduce coa ing complex shapes wi h unables
chemical composi ion o he coa ing [
16
]. Some ime ago, Kokubo [
17
] demons a ed ha an
in i o
chemical-deposi ed bonelike apa i e on cp Ti could be induced by an alkali and hea ea men
p ocess ollowed by a simula ed body luid (SBF) soaking. This apa i e laye does no ha e he
p oblems associa ed wi h he apa i e p oduced by plasma-sp ay echnique; o example, delamina ion
o decomposi ion a high empe a ue [
16
]. Fo NiTi SMA, he e a e ew s udies abou de elopmen o
biomime ic su aces by he mochemical ea men s o imp o e hos issue–implan in eg a ion [
18
,
19
].
Fo example, Chen e al. epo ed an apa i e laye deposi ed on NiTi by ea ing he alloy wi h HNO
3
and hen NaOH aqueous solu ion in SBF [
19
]. They demons a ed ha os eoblas s ac i ely p oli e a ed
on he HA coa ing a six weeks a e implan a ion.
In he p esen s udy, a modi ied he mo-chemical ea men es ablished by Kokubo o inducing
bioac i i y o nickel- i anium has been e alua ed. The i s s ep was ocused on he su ace oxida ion
in o de o dec ease he Ni ion concen a ion, whe eas he second s ep was a he mochemical ea men
wi h NaOH aqueous solu ion o inducing bioac i i y. Finally, he e ec o he supe elas ic/shape
memo y p ope ies in e ms o ans o ma ion empe a u es and c i ical s ess ans o ma ions has
been e alua ed. In e ms o clinical applica ions, he a ia ion o he c i ical s esses implies ha highe
o lowe o ces will be applied o he bone issues. The e o e, o de elopmen o NiTi biomime ic
su aces, hese conside a ions mus be aken in o accoun .
2. Ma e ials and Me hods
TiNi samples aken om comme cial alloys (Memo y Me alle GmbH, Weil am Rhein, Ge many)
designed o shape memo y applica ions wi h ma ensi e phase (Alloy M: 48.5 a % Ni, wi e, ho olled)
and supe elas ici y wi h aus eni e phase (Alloy A: 51.5 a % Ni, hin pla e, annealed) we e selec ed as
objec o he s udy. Samples we e ea ed a 400
˝
C du ing 2.5 h in a low-oxygen p essu e a mosphe e
(3
ˆ
10
´2
mba ) [
20
]. A e wa d, hey we e ea ed wi h 0.5 M NaOH aqueous solu ion a 60
˝
C o
24 h, washed gen ly wi h dis illed wa e and d ied a 40
˝
C o 24 h. Subsequen ly, hey we e hea ed
up o 600
˝
C a a a e o 5
˝
C/min in an elec ic u nace, kep a 600
˝
C o 1 h and hen allowed o
cool in he u nace [
17
]. Finally, he ea ed nickel- i anium subs a es we e soaked in an acellula
simula ed body luid (SBF). I is a solu ion wi h pH = 7.40 and an ion concen a ion nea ly equal o
hose o human blood plasma [
21
]. Each sample was soaked in 40 mL o SBF a 37
˝
C o 2 weeks.
The SBF was enewed e e y wo days.
The c ys allog aphic s udies we e analyzed by G azing-Incidence X- ay Di ac ion (GI-XRD) in
an au oma ic di ac ome e (X’Pe MPD, Philips, Eindho en, The Ne he lands) equipped wi h a hin
ilm a achmen , using Cu-K
α
adia ion. A ange o 2
θ
= 20
˝
–50
˝
was s udied in con inuous mode,
unde a ixed incidence angle o θ = 0.5˝.
The chemical composi ion o he su aces was analyzed by X- ay pho oelec on spec oscopy
(XPS) (SPECS Su ace Nano Analysis GmbH, Be lin, Ge many). Measu emen s we e pe o med using
an XR50 Mg anode sou ce ope a ing a 150 W and a Phoibos 150 MCD-9 de ec o (D8 ad ance, SPECS
Ma e ials 2016,9, 402 3 o 11
Su ace Nano Analysis GmbH, Be lin, Ge many). High esolu ion spec a we e collec ed using 25 eV
a 0.1 eV s eps wi h a chambe p essu e below 7.5
ˆ
10
´9
mba . Binding ene gies we e calib a ed using
he C 1s signal a 480 eV. The XPS dep h p o iles we e eco ded by using a as e ed 3 keV A + ion beam.
The e ching a e is abou 2.3 nm/min. Two specimens we e analyzed o each s udied condi ion.
The ans o ma ion empe a u es (M
s
, M
, A
s
A
) o he alloy ob ained we e de e mined by
Di e en ial Scanning Calo ime y (DSC, 2920 modula ed DSC, TA Ins umen s, New Cas le, DE, USA)
wi h a hea ing amp o 10 ˝C/min and a cooling amp o 2 ˝C/min.
Tensile es s we e ca ied ou on a se o-hyd aulic es ing machine (MTS-Bionix 858, Eden P ai ie,
MN, USA), wo king a a c oss-head speed o 10 mm/min. Fi e NiTi specimens es ed we e cylinde s
o 0.457 mm in diame e and o 45 mm in leng h. The c i ical s esses (aus eni e o s ess-induced
ma ensi e) we e de e mined om hese es s, which we e pe o med in simula ed body luid a 37
˝
C.
Fo he nickel ion elease, samples we e imme sed in 6 mL o con en ional SBF a pH = 7.4, 37
˝
C
un il 30 days. A Day 5, SBF was enewed o a oid sa u a ion o he medium. The Ni eleased was
quan i ied by G aphi e Fu nace A omic Abso p ion Spec oscopy (GFAAS) (Ewai, Beijing, China) a 1
and 5 h, and 1, 2, 5, 15 and 30 days. The esul s we e no malized by he speci ic su ace a ea o each
sample and a e mean alues o h ee measu emen s.
Fo he
in i o
s udies, human os eoblas -like MG63 cells (Ame ican Type Cul u e Collec ion,
Rock ille, MD, USA) we e g own in Dulbecco’s modi ied Eagle’s medium (DMEM, Gibco, Paisley,
UK) supplemen ed wi h 10% e al cal se um (FCS), 1% penicillin/s ep omycin, 1% L-glu amine, 1%
py u a e (Sigma, S . Louis, MO, USA) a 37
˝
C in 5% CO
2
/95% ai a mosphe e and 100% humidi y.
Fo he expe imen s, cells we e ha es ed a 70%–90% con luence by T ipsin/EDTA, cen i uged and
e-suspended in se um- ee medium be o e pla ing on he samples. Samples we e placed in o a 24-well
issue cul u e pla es (TCPS) (Falcon, Bec on Dickinson & Company, Mad id, Spain) hen 3
ˆ
10
4
cells
pe sample we e added and incuba ed o 2 h in se um ee medium. To obse e cell adhesion and
he o e all mo phology o adhe ing cells, he samples we e s ained wi h luo escein diace a e (FDA)
added di ec ly o he medium o gi e a inal concen a ion o 1
µ
g/mL ( om a s ock o 1 mg/mL
in ace one) o 3 min. The s ained li ing cells, he only ones able o con e he die o a luo escen
analog, we e iewed and pho og aphed wi h an in e ed luo escen mic oscope (Nikon, Eclipse E600,
Ba celona, Spain) using he g een channel.
Fo he
in i o
s udies, wel e emale adul New Zealand Whi e (NZW) abbi s (3.5 kg b.w.,
Cha les Ri e , Sain Aubin les Elboeu , F ance) we e ope a ed on unde gene al anes hesia, pe o med
by in amuscula injec ions o xylazine (5 mg/kg) and ke amine (35 mg/kg). A e la e al bila e al
knee a h o omy, a d illed bone de ec o 4 mm in diame e and 6 mm deep was cen e ed on he
la e al condyle. All animal handling and su gical p ocedu es we e conduc ed acco ding o Eu opean
Communi y guidelines o he ca e and use o labo a o y animals (DE 86/609/CEE) and app o ed by
he local e e ina y school e hical commi ee.
Fo he his ological analysis, emo al condyles we e ha es ed and pe iphe al so issue was
emo ed. Specimens we e ixed o 7 days in 4% o maldehyde neu al solu ion insed in wa e ,
dehyd a ed in g aded se ies o e hanol ( om 70% o 100%) and embedded in polyme hyl me hac yla e.
The pe cen age o bone implan con ac (BIC) was calcula ed using a semi-au oma ic bina y ea men
on each image. Quali a i e examina ions we e pe o med by ligh mic oscopy on s ained sec ions
(1% me hylenblue and 0.3% basic uchsin).
Fo s a is ical analysis, all da a we e analyzed by a nonpa ame ic U Mann-Whi ney es (IBM SPSS
S a is ics 20 so wa e, A monk, NY, USA). S a is ical signi icance was se a a P alue < 0.05.
3. Expe imen al Resul s
Dep h p o iling o he O
2
-oxidized Ti was pe o med by cu e i ing o he Ti 2p spec um, which
ga e an es ima ion o he con ibu ion o he TiO
2
oxide in he o al i anium signal (Table 1). Ti 2p
spec um was decon olu ed in o eigh peaks co esponding o ou chemical s a es om double
consis ing o Ti 2p
1/2
and Ti 2p
3/2
(spec a no shown): wo s ong Ti
4+
peaks, i.e., 2p
1/2
(Ti
4+
, 464.6 eV)
Ma e ials 2016,9, 402 4 o 11
and 2p
3/2
oxide (Ti
4+
, 458.8 eV), wo Ti
3+
peaks (457 and 463.1 eV) and wo Ti
2+
peaks (455.3 and
461.7 eV), as well as wo me allic Ti peaks (454.3 and 460.5 eV). The high- esolu ion Ti 2p XPS spec a
we e compa able o hose ob ained by o he au ho s [22,23].
Table 1.
Va ia ion o he i anium oxide composi ion as a unc ion o dep h o di e en su ace
ea men s s udied (UT: un ea ed samples; OT: ea ed samples).
T ea men Spu e ing Time (min)/Dep h (nm) Chemical Elemen % o o al Ti 2p Signal
UT 0/0 Ti0Ti+2, Ti+3/Ti+4 8/10/82
OT 0/0 Ti+4 100
OT 1/6 Ti3+ Ti4+ 61/39
Fo un ea ed samples, he Ti 2p pho opeaks ob ained indica e he p esence o a s oichiome ic
TiO
2
, as well as Ti
0
signals indica ing ha p o ec i e oxide laye hickness is e y hin (below he dep h
limi o XPS). Howe e , o he oxidized samples, i can clea ly be obse ed ha Ti is mos ly p esen as
Ti
4+
and he Ti
0
signal is p ac ically negligible showing ha he oxide laye has g own. Dep h p o iling
e eals ha he Ti
+4
and Ti
+3
species comp ise he ou e mos o he oxide laye ; he emaining oxide
laye , close o he me al–oxide in e ace, consis s o Ti
+2
and Ti
0
Finally, he dep h p o iling o he Ni as
a unc ion o spu e ing ime is plo ed in Figu e 1, whe e a zone deple ed in Ni (<3 a %) is o med up
o app oxima ely 25 nm in dep h. I is expec ed ha he p esence o a ela i ely hicke TiO
2
laye , ee
o nickel, could p e en s he nickel ions elease o he su ounding issues.
Ma e ials 2016, 9, 402 4 o 10
Table 1. Va ia ion o he i anium oxide composi ion as a unc ion o dep h o di e en su ace
ea men s s udied (UT: un ea ed samples; OT: ea ed samples).
T ea men Spu e ing Time (min)
/
Dep h (nm) Chemical Elemen % o o al Ti 2p Signal
UT 0/0 Ti0 Ti+2, Ti+3/Ti+4 8/10/82
OT 0/0 Ti+4 100
OT 1/6 Ti3+ Ti4+ 61/39
Fo un ea ed samples, he Ti 2p pho opeaks ob ained indica e he p esence o a s oichiome ic
TiO
2
, as well as Ti
0
signals indica ing ha p o ec i e oxide laye hickness is e y hin (below he
dep h limi o XPS). Howe e , o he oxidized samples, i can clea ly be obse ed ha Ti is mos ly
p esen as Ti
4+
and he Ti
0
signal is p ac ically negligible showing ha he oxide laye has g own.
Dep h p o iling e eals ha he Ti
+4
and Ti
+3
species comp ise he ou e mos o he oxide laye ; he
emaining oxide laye , close o he me al–oxide in e ace, consis s o Ti
+2
and Ti
0
Finally, he dep h
p o iling o he Ni as a unc ion o spu e ing ime is plo ed in Figu e 1, whe e a zone deple ed in Ni
(<3 a %) is o med up o app oxima ely 25 nm in dep h. I is expec ed ha he p esence o a ela i ely
hicke TiO
2
laye , ee o nickel, could p e en s he nickel ions elease o he su ounding issues.
Figu e 1. XPS dep h p o ile o he nickel o aus eni ic alloy, a e oxida ion ea men .
Figu e 2 shows he appea ance o he samples ea ed wi h NaOH solu ion and hea ed up o
600 °C. A mic opo ous laye made up o an alkaline i ana e has been induced on he NiTi su ace.
The mo phology ob ained is e y simila o o he s udies o biomime ic su aces ea men s
pe o med on i anium [24]. The co esponding GI-XRD pa e ns a e e y simila o bo h NiTi alloys
(aus en ic and ma ensi ic) showing peaks co esponding o me allic nickel- i anium, u ile and
di e en sodium- i ana es s oichiome ies, o example, Na
2
Ti
3
O
7
(JCPDS #72-0148), Na
2
Ti
6
O
13
(JCPDS #73-1398) and NaTiO
2
(JCPDS #89-2784). Finally, when he alkaline i ana e hyd ogel su aces
we e imme sed in SBF a 37 °C o a pe iod o ime as sho as h ee days, an apa i e laye appea ed
(Figu e 3). The mo phology ob ained was consis en wi h hose epo ed in he li e a u e [25] and he
na u e o his bioac i e laye was con i med by GI-X- ay di ac ome e . I showed peaks
co esponding o apa i e c ys als wi h almos non-exis en alkaline i ana es peaks (Figu e 3a). The
XRD peaks o apa i e nea 32° and 26° a e e iden .
Figu e 1. XPS dep h p o ile o he nickel o aus eni ic alloy, a e oxida ion ea men .
Figu e 2shows he appea ance o he samples ea ed wi h NaOH solu ion and hea ed up
o 600
˝
C. A mic opo ous laye made up o an alkaline i ana e has been induced on he NiTi
su ace. The mo phology ob ained is e y simila o o he s udies o biomime ic su aces ea men s
pe o med on i anium [
24
]. The co esponding GI-XRD pa e ns a e e y simila o bo h NiTi
alloys (aus en ic and ma ensi ic) showing peaks co esponding o me allic nickel- i anium, u ile
and di e en sodium- i ana es s oichiome ies, o example, Na
2
Ti
3
O
7
(JCPDS #72-0148), Na
2
Ti
6
O
13
(JCPDS #73-1398) and NaTiO
2
(JCPDS #89-2784). Finally, when he alkaline i ana e hyd ogel su aces
we e imme sed in SBF a 37
˝
C o a pe iod o ime as sho as h ee days, an apa i e laye appea ed
(Figu e 3). The mo phology ob ained was consis en wi h hose epo ed in he li e a u e [
25
] and he
na u e o his bioac i e laye was con i med by GI-X- ay di ac ome e . I showed peaks co esponding
Ma e ials 2016,9, 402 5 o 11
o apa i e c ys als wi h almos non-exis en alkaline i ana es peaks (Figu e 3a). The XRD peaks o
apa i e nea 32˝and 26˝a e e iden .
Ma e ials 2016, 9, 402 5 o 10
(a) (b)
Figu e 2. (a) SEM mic og aph showing a mic opo ous laye o sodium i ana e on NiTi su ace
(alloy A); and (b) GI-XRD pa e n o sodium i ana e laye .
(a) (b)
Figu e 3. (a) SEM mic og aph o apa i e laye on NiTi su ace (Alloy A) a e soaked in SBF o h ee
days; and (b) GI-XRD pa e n showing peaks co esponding o he apa i e c ys als (32° and 26°).
Ni ions elease concen a ion as a unc ion o imme sion ime in SBF a 37 °C was e alua ed. The
expe imen al esul s showed ha he Ni ion elease is e icien ly educed and he highes Ni elease
occu s wi hin he i s hou . A e 24 h o imme sion, i was negligible. The o al Ni elease a e one
mon h o imme sion was abou 100 ± 32, 32 ± 5, and 10 ± 4 ng/cm2 o con ol, oxidized, and bioac i e
su aces, espec i ely. This co esponds o espec i e mean alues o 0.29, 0.13 and 0.04 μg/mL in ou
sys em.
The su ace modi ica ion p oposed induced highe cell ec ui men and cell sp eading a e only
2 h o cul u e (Figu e 4a). Simila ly, cell sp eading quan i ica ion p esen ed conside ably highe a ea
alues o he bioac i e NiTi (1350 ± 240 μm2), and no di e ences we e obse ed among he o he
samples. Immuno luo escence images showed ha cells on un ea ed NiTi su ace exhibi ed a
la ened and less-ex ended mo phology sp ead han hose obse ed in he bioac i e NiTi (Figu e
4b). La ge cy oplasmic ex ensions like ilopodia ancho ed he cell o he subs a e and do sal ac i i y
ha e been obse ed in he os eoblas cul u ed on he bioac i e su ace in ela ion o o he s samples.
Figu e 2.
(
a
) SEM mic og aph showing a mic opo ous laye o sodium i ana e on NiTi su ace (alloy A);
and (b) GI-XRD pa e n o sodium i ana e laye .
Ma e ials 2016, 9, 402 5 o 10
(a) (b)
Figu e 2. (a) SEM mic og aph showing a mic opo ous laye o sodium i ana e on NiTi su ace
(alloy A); and (b) GI-XRD pa e n o sodium i ana e laye .
(a) (b)
Figu e 3. (a) SEM mic og aph o apa i e laye on NiTi su ace (Alloy A) a e soaked in SBF o h ee
days; and (b) GI-XRD pa e n showing peaks co esponding o he apa i e c ys als (32° and 26°).
Ni ions elease concen a ion as a unc ion o imme sion ime in SBF a 37 °C was e alua ed. The
expe imen al esul s showed ha he Ni ion elease is e icien ly educed and he highes Ni elease
occu s wi hin he i s hou . A e 24 h o imme sion, i was negligible. The o al Ni elease a e one
mon h o imme sion was abou 100 ± 32, 32 ± 5, and 10 ± 4 ng/cm2 o con ol, oxidized, and bioac i e
su aces, espec i ely. This co esponds o espec i e mean alues o 0.29, 0.13 and 0.04 μg/mL in ou
sys em.
The su ace modi ica ion p oposed induced highe cell ec ui men and cell sp eading a e only
2 h o cul u e (Figu e 4a). Simila ly, cell sp eading quan i ica ion p esen ed conside ably highe a ea
alues o he bioac i e NiTi (1350 ± 240 μm2), and no di e ences we e obse ed among he o he
samples. Immuno luo escence images showed ha cells on un ea ed NiTi su ace exhibi ed a
la ened and less-ex ended mo phology sp ead han hose obse ed in he bioac i e NiTi (Figu e
4b). La ge cy oplasmic ex ensions like ilopodia ancho ed he cell o he subs a e and do sal ac i i y
ha e been obse ed in he os eoblas cul u ed on he bioac i e su ace in ela ion o o he s samples.
Figu e 3.
(
a
) SEM mic og aph o apa i e laye on NiTi su ace (Alloy A) a e soaked in SBF o
h ee days; and (
b
) GI-XRD pa e n showing peaks co esponding o he apa i e c ys als (32
˝
and 26
˝
).
Ni ions elease concen a ion as a unc ion o imme sion ime in SBF a 37
˝
C was e alua ed.
The expe imen al esul s showed ha he Ni ion elease is e icien ly educed and he highes Ni
elease occu s wi hin he i s hou . A e 24 h o imme sion, i was negligible. The o al Ni elease
a e one mon h o imme sion was abou 100
˘
32, 32
˘
5, and 10
˘
4 ng/cm
2
o con ol, oxidized,
and bioac i e su aces, espec i ely. This co esponds o espec i e mean alues o 0.29, 0.13 and
0.04 µg/mL in ou sys em.
The su ace modi ica ion p oposed induced highe cell ec ui men and cell sp eading a e only
2 h o cul u e (Figu e 4a). Simila ly, cell sp eading quan i ica ion p esen ed conside ably highe
a ea alues o he bioac i e NiTi (1350
˘
240
µ
m
2
), and no di e ences we e obse ed among he
o he samples. Immuno luo escence images showed ha cells on un ea ed NiTi su ace exhibi ed
a la ened and less-ex ended mo phology sp ead han hose obse ed in he bioac i e NiTi (Figu e 4b).

Ma e ials 2016,9, 402 6 o 11
La ge cy oplasmic ex ensions like ilopodia ancho ed he cell o he subs a e and do sal ac i i y ha e
been obse ed in he os eoblas cul u ed on he bioac i e su ace in ela ion o o he s samples.
Ma e ials 2016, 9, 402 6 o 10
(a)
(b)
Figu e 4. (a) Cell sp eading and cell numbe a e 2 h o cul u e co esponding o di e en su aces:
C, Te lon; UT, un ea ed Ti; OX, oxidized Ti; and BI, bioac i e Ti; (b) Rep esen a i e luo escence
mic oscopy images o MG63 cells on un ea ed NiTi (A) and bioac i e NiTi (B) a e se en days o
incuba ion (alloy A).
Finally, Table 2 summa izes he pe cen age o he bone implan con ac (BIC) a mic oscopic
le el o un ea ed and bioac i e implan s, espec i ely. The con ol NiTi implan s had lowe
pe cen ages o di ec con ac bone wi h s a is ically signi ican di e ences (Fishe ’s es ; 0.05) han he
he mochemical ea ed samples. A e one week, BIC alues we e signi ican ly highe o he
bioac i e implan s, anging be ween 22% and 32%, while he mean alues o he un ea ed NiTi
implan s anged be ween 5% and 13%. This means ha an osseoin eg a ion is achie ed o bioac i e
implan s a imes o implan a ion as b ie as 1 week. O e all, he specimens showed he p esence o
emodeling ac i i y in he bone immedia ely adjacen o he mic oimplan . The newly- o med bone
showed ea ly s ages o ma u ing and emodeling, pa icula ly on he ea ed su ace (Figu e 5).
Table 2. Bone index con ac a e one and six weeks o implan a ion (columns) in pe cen .
Type o Implan s 1 Week 6 Weeks
NiTi 9% (±4%) 44% (±11%)
NiTi bioac i e 27% (±5%) 68% (±15%)
Figu e 4.
(
a
) Cell sp eading and cell numbe a e 2 h o cul u e co esponding o di e en su aces:
C
, Te lon;
UT
, un ea ed Ti;
OX
, oxidized Ti; and
BI
, bioac i e Ti; (
b
) Rep esen a i e luo escence
mic oscopy images o MG63 cells on un ea ed NiTi (
A
) and bioac i e NiTi (
B
) a e se en days o
incuba ion (alloy A).
Finally, Table 2summa izes he pe cen age o he bone implan con ac (BIC) a mic oscopic le el
o un ea ed and bioac i e implan s, espec i ely. The con ol NiTi implan s had lowe pe cen ages o
di ec con ac bone wi h s a is ically signi ican di e ences (Fishe ’s es ; 0.05) han he he mochemical
ea ed samples. A e one week, BIC alues we e signi ican ly highe o he bioac i e implan s,
anging be ween 22% and 32%, while he mean alues o he un ea ed NiTi implan s anged be ween
5% and 13%. This means ha an osseoin eg a ion is achie ed o bioac i e implan s a imes o
implan a ion as b ie as 1 week. O e all, he specimens showed he p esence o emodeling ac i i y in
he bone immedia ely adjacen o he mic oimplan . The newly- o med bone showed ea ly s ages o
ma u ing and emodeling, pa icula ly on he ea ed su ace (Figu e 5).
Table 2. Bone index con ac a e one and six weeks o implan a ion (columns) in pe cen .
Type o Implan s 1 Week 6 Weeks
NiTi 9% (˘4%) 44% (˘11%)
NiTi bioac i e 27% (˘5%) 68% (˘15%)
Ma e ials 2016,9, 402 7 o 11
Ma e ials 2016, 9, 402 7 o 10
Figu e 5. His ological mic og aph showing he in e ace bone–implan o he bioac i e NiTi alloy
(×400). The implan –bone in e ace is almos comple ely co e ed a e six weeks o implan a ion.
Finally, Table 3 shows he ans o ma ion empe a u es (A
s
, A
, M
s
, and M
) o un ea ed and
bioac i es NiTi alloys. The DSC cu es a di e en cooling/hea ing cycles do no shown di e ences
and R-phase peaks we e no obse ed. Fo bo h mic os uc u es (aus eni e/ma ensi e), a dec ease o
he ans o ma ion empe a u es was obse ed due o he he mochemical ea men pe o med.
Mo eo e , he c i ical s esses equi ed o induce he s ess-induced ma ensi e, σ
β
→
M
o σ
M
→
β
a
20 °C and 37 °C, espec i ely, ha e been changed. The ans o ma ion s esses o he au eni ic NiTi
alloy a e shown in Table 4. I is clea ly shown ha highe s esses we e equi ed o ans o m he
aus eni e o s ess-induced ma ensi e and ice e sa in he ea ed samples.
Table 3. T ans o ma ion empe a u es (°C) o he Ni-Ti s udied.
Alloy M
s
M
A
s
A
M 74.1 ± 0.3 55.7 ± 0.4 86.9 ± 0.1 129.3 ± 0.7
M-bioac i e 64.2 ± 0.6 46.1 ± 0.5 71.0 ± 0.1 109.3 ± 0.7
A 9.8 ± 0.2 6.2 ± 0.3 7.1 ± 0.4 20.4 ± 0.5
A-bioac i e 0.5 ± 2.4 −3.9 ± 1.2 −0.4 ± 1.7 11.2 ± 4.5
Table 4. C i ical s esses a di e en es empe a u es o aus eni ic NiTi alloy.
Au eni ic NiTi Alloy C i ical S esses 20 °C 37 °C
NiTi o iginal σ
β
→
SIM
(MPa) 150 ± 17 262 ± 24
σ
SIM
→
β
(MPa) 55 ± 5 211 ± 19
NiTi bioac i e σ
β
→
SIM
(MPa) 180 ± 15 290 ± 22
σ
SIM
→
β
(MPa) 65 ± 9 183 ± 19
4. Discussion
In he i s s ep, a hicke p o ec i e laye o u ile/anas ase, ee o nickel, has been p oduced on
he NiTi su aces. The oxida ion p ocess a low oxygen p essu e allowed a dec ease o Ni
concen a ion on he su ace (Figu e 1). In he li e a u e, s udies o NiTi oxida ion wi h simila esul s
exis [22,23,26,27]. Fo example, ou indings a e simila o hose ob ained by Chan e al. [28] using an
oxida ion ea men . Su ace oxida ion p oduced a smoo h p o ec i e nickel- ee oxide laye wi h a
ela i ely small amoun o Ni species a he ai /oxide in e ace. S udies published in he li e a u e
could be es ablished o explain hese esul s [20]. Acco ding o A mi age e al. [29], he oxide laye is
p edominan ly o med by inwa d di usion o O ions inside NiTi la ice. Because ΔG
o ma ion (298K)
o
TiO
2
and NiO a e −212.6 kcal∙mol
−1
and −50.6 kcal∙mol
−1
, espec i ely [30,31], he e is a p e e en ial
oxida ion o Ti. Ni emains in i s me allic s a e. The o ma ion o s oichiome ic TiO
2
is p og essi e
and TiO
x
(x < 4) may be p esen unde nea h [23] a he TiO
2
-NiTi in e ace. In ha si ua ion, Espinós
e al. [32] showed ha Ni a oms in me allic s a e di use easily h ough de ec i e TiO
x
(x < 4). The e is
Figu e 5.
His ological mic og aph showing he in e ace bone–implan o he bioac i e NiTi alloy
(ˆ400). The implan –bone in e ace is almos comple ely co e ed a e six weeks o implan a ion.
Finally, Table 3shows he ans o ma ion empe a u es (A
s
, A
, M
s
, and M
) o un ea ed and
bioac i es NiTi alloys. The DSC cu es a di e en cooling/hea ing cycles do no shown di e ences
and R-phase peaks we e no obse ed. Fo bo h mic os uc u es (aus eni e/ma ensi e), a dec ease
o he ans o ma ion empe a u es was obse ed due o he he mochemical ea men pe o med.
Mo eo e , he c i ical s esses equi ed o induce he s ess-induced ma ensi e,
σβÑM
o
σMÑβ
a
20
˝
C and 37
˝
C, espec i ely, ha e been changed. The ans o ma ion s esses o he au eni ic NiTi
alloy a e shown in Table 4. I is clea ly shown ha highe s esses we e equi ed o ans o m he
aus eni e o s ess-induced ma ensi e and ice e sa in he ea ed samples.
Table 3. T ans o ma ion empe a u es (˝C) o he Ni-Ti s udied.
Alloy MsM AsA
M 74.1 ˘0.3 55.7 ˘0.4 86.9 ˘0.1 129.3 ˘0.7
M-bioac i e 64.2 ˘0.6 46.1 ˘0.5 71.0 ˘0.1 109.3 ˘0.7
A 9.8 ˘0.2 6.2 ˘0.3 7.1 ˘0.4 20.4 ˘0.5
A-bioac i e 0.5 ˘2.4 ´3.9 ˘1.2 ´0.4 ˘1.7 11.2 ˘4.5
Table 4. C i ical s esses a di e en es empe a u es o aus eni ic NiTi alloy.
Au eni ic NiTi Alloy C i ical S esses 20 ˝C 37 ˝C
NiTi o iginal σβÑSIM (MPa) 150 ˘17 262 ˘24
σSIMÑβ(MPa) 55 ˘5 211 ˘19
NiTi bioac i e σβÑSIM (MPa) 180 ˘15 290 ˘22
σSIMÑβ(MPa) 65 ˘9 183 ˘19
4. Discussion
In he i s s ep, a hicke p o ec i e laye o u ile/anas ase, ee o nickel, has been p oduced
on he NiTi su aces. The oxida ion p ocess a low oxygen p essu e allowed a dec ease o Ni
concen a ion on he su ace (Figu e 1). In he li e a u e, s udies o NiTi oxida ion wi h simila esul s
exis [
22
,
23
,
26
,
27
]. Fo example, ou indings a e simila o hose ob ained by Chan e al. [
28
] using
an oxida ion ea men . Su ace oxida ion p oduced a smoo h p o ec i e nickel- ee oxide laye wi h
a ela i ely small amoun o Ni species a he ai /oxide in e ace. S udies published in he li e a u e
could be es ablished o explain hese esul s [
20
]. Acco ding o A mi age e al. [
29
], he oxide laye is
p edominan ly o med by inwa d di usion o O ions inside NiTi la ice. Because
∆
G
o ma ion (298K)
o
Ma e ials 2016,9, 402 8 o 11
TiO
2
and NiO a e
´
212.6 kcal
¨
mol
´1
and
´
50.6 kcal
¨
mol
´1
, espec i ely [
30
,
31
], he e is a p e e en ial
oxida ion o Ti. Ni emains in i s me allic s a e. The o ma ion o s oichiome ic TiO
2
is p og essi e
and TiO
x
(x< 4) may be p esen unde nea h [
23
] a he TiO
2
-NiTi in e ace. In ha si ua ion,
Espinós e al. [32] showed ha Ni a oms in me allic s a e di use easily h ough de ec i e TiOx(x< 4).
The e is a mig a ion o Ni a oms leading o i s disappea ance om he su ace. This could explain he
Ni-en iched laye obse ed below he Ni-deple ed zone in he concen a ion p o iles ob ained.
In he second s ep, he o ma ion o a sodium alkaline i ana e hyd ogel om oxidized su ace
has been induced (Figu e 2). I is epo ed ha he apa i e o ming abili y is dependen on he i ania
c ys alline s uc u e. The i ania gels wi h amo phous s uc u e did no induce apa i e o ma ion on
hei su aces in SBF, whe eas i ania wi h anas ase/ u ile s uc u e induced apa i e o ma ion [
33
].
In he p esen s udy, GI-XRD s udies ha e shown a c ys allog aphic laye composed by u ile as
well as di e en sodium i ana es c ys als (Figu e 2). Mo eo e , he simula ed body luid (SBF)
co obo a ed he abili y o induce apa i e o ma ion (Figu e 3). I is well known ha he SBF, which
has a simila ion concen a ions as hose o he human blood plasma, could ep oduce
in i o
su ace
changes [
34
]. The good esul s ob ained be o e (Figu e 3) a e co obo a ed
in i o
and
in i o
cells
s udies. The bioac i i y o he NiTi modi ied su ace is imp o ed (Figu es 4and 5). Bone implan
con ac alues indica ed ha he osseoin eg a ion p ocess is imp o ed (Table 3).
The elease o Ni ions can be ha m ul o he cells o he adjacen issues and i is closely linked o
he co osion beha io . They a e impo an pa ame e s o ake in o accoun o medical de ices as hey
can ha e long- e m
in i o
implica ions [
7
,
8
]. The e a e s udies showing ha Ni ion elease is able o
induce alle gic esponse [
35
–
38
]. Fo example, Cede b an e al. [
39
] showed ha e en a quan i y as
small as 1.2
µ
g/mL o Ni could induce an inc ease in lymphocy es p oli e a ion and IL10 sec e ion in
subjec s alle gic o Ni. Thus, he esul s ob ained o ou bioac i e su aces (0.05
µ
g/mL) migh be o
g ea impo ance o imp o e he long- e m biocompa ibili y p ope ies.
Nume ous s udies on he de elopmen o new biomime ic su aces on NiTi alloys a e epo ed;
howe e , hey do no pay a en ion o how he pseudoelas ic o shape memo y p ope ies could be
a ec ed by hese ea men s. Fo example, Table 3clea ly shows ha highe s esses we e equi ed o
ans o m he aus eni e o s ess-induced ma ensi e and ice e sa in he ea ed samples. These highe
s esses a e, p obably, due o he ans o ma ion empe a u e (M
s
) was a om 37
˝
C o ea ed
samples (Table 3). I is no clea why he ans o ma ion empe a u es (A
s
, A
, M
s
, and M
) dec ease
due o he he mo-chemical ea men . One hypo hesis could be he o ma ion o he i anium- ich
p ecipi a es in o he NiTi bulk dec easing he i anium con en du ing he he mochemical ea men .
Fo example, A ciniegas e al. [
37
] ha e demons a ed he appea ance o NiTi p ecipi a es ich in
Ti anium (Ti
2
Ni) when a NiTi alloy (50 a % wi h ma ensi ic mic os uc u e) a e subjec ed a simila
hea ea men s. Ti
2
Ni p ecipi a es impeded he g ow h o ma ensi ic pla es and can be nuclea ion
sou ce o hem. Simila obse a ions we e epo ed by Fi s o [
23
] whe e he ma ensi ic ans o ma ion
o he subs a e NiTi alloy benea h he oxidized scale is supp essed below oom empe a u e because
o he deple ion o he NiTi la ice by Ti which is consumed in he oxidized scale o p oduce Ti oxides.
Howe e , u he s udies by TEM will be ca ied ou in he u u e.
Highe s ess was needed o ans o m he aus eni e o s ess-induced ma ensi e in he bioac i e
samples because he ans o ma ion empe a u e was a om 37
˝
C han in he o iginal samples.
In e ms o clinical applica ions, his small inc ease o he c i ical s esses (app oxima ely 18 MPa)
implies ha highe o ces will be applied o he bone issues. The elas ic de o ma ion o a NiTi sample
and he subsequen elease o i s elas ic ene gy o e a pe iod o ime inc ease he loads on he li ing
issue. I is gene ally assumed ha op imal load ans e in he li ing issues is achie ed by applying
o ces ha a e low in magni ude and con inuous in na u e [
40
,
41
]. Fo una ely, he he mochemical
me hod p oposed does no p oduce la ge a ia ions, bu his is a ac ha mus be aken in o accoun
in e ms o clinical applica ions.
Ma e ials 2016,9, 402 9 o 11
5. Conclusions
A new he mo-chemical ea men o inducing bioac i i y on se e al NiTi alloys has been
p oposed. In he i s s ep, a sa e ba ie agains Ni elease has been p oduced on he su ace by
means o a hicke u ile/anas ase p o ec i e laye ee o nickel. In he second s ep, a sodium alkaline
i ana e hyd ogel, which has he abili y o induce apa i e o ma ion, has been pe o med om oxidized
su ace. The bioac i i y o he ea ed NiTi alloys has been co obo a ed wi h bo h
in i o
and
in i o
s udies. Mo eo e , he imp o emen o he hos issue–implan in eg a ion in e ms o Ni ions elease
and co osion esis ance has been demons a ed. Howe e , he ans o ma ion empe a u es (A
s
, A
,
M
s
, and M
), as well as he c i ical s esses (
σβôM
), ha e been sligh ly modi ied due o he su ace
modi ica ion. In e ms o clinical applica ions, he a ia ion o he c i ical s esses implies ha highe
o lowe o ces will be applied o he bone issues.
Acknowledgmen s:
This wo k was suppo ed by cicy Spanish p ojec MAT2015-67183-R and he Ando a and
Ca alan Go e nmen o CTP g an s.
Au ho Con ibu ions:
F.J. Gil p oposed he esea ch opic and supe ised he en i e p ojec along wi h
Luis-Albe o B a o-González and Edua do Espina . Elisa Rupé ez and José Ma ía Mane o designed he
expe imen s, pe o med he physicochemical cha ac e iza ion as well as he biological expe imen s, and w o e he
manusc ip . All he au ho s con ibu ed o he w i ing and app o ed he inal manusc ip .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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