

Ci a ion: C uz, N.; Gil, J.; Punse , M.;
Mane o, J.M.; Tondela, J.P.; Ve degue ,
P.; Apa icio, C.; Rúpe ez, E. Rele an
Aspec s o Pi anha Passi a ion in
Ti6Al4V Alloy Den al Meshes. Coa ings
2022,12, 154. h ps://doi.o g/
10.3390/coa ings12020154
Academic Edi o s: Jasmina P imožiˇc
and Vincenzo D’An ò
Recei ed: 28 Decembe 2021
Accep ed: 24 Janua y 2022
Published: 27 Janua y 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
coa ings
A icle
Rele an Aspec s o Pi anha Passi a ion in Ti6Al4V Alloy
Den al Meshes
Nuno C uz 1, Ja ie Gil 1,2,* , Miquel Punse 3,4,5,6 , JoséMa ía Mane o 3,4,6, João Paulo Tondela 7,* ,
Pablo Ve degue 1, Con ado Apa icio 2and Elisa Rúpe ez 3,4,6
1Bioenginee ing Ins i u e o Technology, In e na ional Uni e si y o Ca alonia (UIC), 08195 Ba celona, Spain;
[email p o ec ed] (N.C.); pablo e [email p o ec ed] (P.V.)
2School o Den is y, In e na ional Uni e si y o Ca alonia (UIC), 08195 Ba celona, Spain; [email p o ec ed]
3Bioma e ials, Biomechanics and Tissue Enginee ing G oup (BBT), Depa men o Ma e ials Science and
Enginee ing, Technical Uni e si y o Ca alonia (UPC), 08019 Ba celona, Spain; [email p o ec ed] (M.P.);
[email p o ec ed] (J.M.M.); elisa. [email p o ec ed] (E.R.)
4Ba celona Resea ch Cen e in Mul iscale Science and Enginee ing, Technical Uni e si y o Ca alonia (UPC),
08019 Ba celona, Spain
5
Inno a ion and Technology Cen e (CIT), Poly echnic Uni e si y o Ca alonia (UPC), 08034 Ba celona, Spain
6San Joan de Déu Resea ch Ins i u e (IRSJD), 08034 Ba celona, Spain
7CIROS om he Facul y o Medicine, Uni e si y o Coimb a, FMUC, 3004-531 Coimb a, Po ugal
*Co espondence: xa ie [email p o ec ed] (J.G.); [email p o ec ed] (J.P.T.)
Abs ac :
Passi a ion o i anium alloy den al meshes cleans hei su ace and o ms a hin laye o
p o ec i e oxide (TiO
2
) on he su ace o he ma e ial o imp o e esis ance o co osion and p e en
elease o ions o he physiological en i onmen . The mos common chemical agen o he passi a-
ion p ocess o i anium meshes is hyd ochlo ic acid (HCl). In his wo k, we in oduce he use o
Pi anha solu ion (H
2
SO
4
and H
2
O
2
) as a passi a ing and bac e icidal agen o me allic den al meshes.
Meshes o g ade 5 i anium alloy (Ti6Al4V) we e es ed a e di e en ea men s: as- ecei ed con ol
(C ), passi a ed by HCl, and passi a ed by Pi anha solu ion. Physical-chemical cha ac e iza ion o
all ea ed su aces was ca ied ou by scanning elec on mic oscopy (SEM), con ocal mic oscopy and
sessile d op goniome y o assess meshes’ opog aphy, elemen al composi ion, oughness, we abili y
and su ace ee ene gy, ha is, ele an p ope ies wi h po en ial e ec s o he biological esponse
o he ma e ial. Mo eo e , open ci cui po en ial and po en iodynamic es s we e ca ied ou o
e alua e he co osion beha io o he di e en ly- ea ed meshes unde physiological condi ions.
Ion elease es s we e conduc ed using Induc i ely Coupled Plasma mass spec ome y (ICP-MS).
The an ibac e ial ac i i y by p e en ion o bac e ial adhesion es s on he meshes was pe o med o
wo di e en bac e ial s ains, Pseudomonas ae uginosa (G am-) and S ep ococcus sanguinis (G am+).
Addi ionally, a bac e ial iabili y s udy was pe o med wi h he LIVE/DEAD es . We complemen ed
he an ibac e ial s udy by coun ing cells a ached o he su ace o he meshes isualized by SEM.
Ou esul s showed ha he passi a ion o i anium meshes wi h Pi anha solu ion imp o ed hei
hyd ophilici y and con e ed a no ably highe bac e icidal ac i i y in compa ison wi h he meshes
passi a ed wi h HCl. This unique esponse can be a ibu ed o di e ences in he ob ained nano ex-
u es o he TiO
2
laye . Howe e , Pi anha solu ion ea men dec eased elec ochemical s abili y
and inc eased ion elease as a esul o he po ous coa ing o med on he ea ed su aces, which
can comp omise hei co osion esis ance. F amed by he limi a ions o his wo k, we conclude
ha using Pi anha solu ion is a iable al e na i e me hod o passi a ing i anium den al meshes
wi h bene icial an ibac e ial p ope ies ha me i s u he alida ion o i s ansla ion as a ea men
applied o clinically-used meshes.
Keywo ds:
i anium; den al meshes; passi a ion; pi anha; co osion esis ance; ion elease; bac e ial
adhesion
Coa ings 2022,12, 154. h ps://doi.o g/10.3390/coa ings12020154 h ps://www.mdpi.com/jou nal/coa ings
Coa ings 2022,12, 154 2 o 17
1. In oduc ion
The amoun o bone is pa amoun o p edic ably achie e success and long- e m
su i al o implan -suppo ed ehabili a ions. Ac ually, implan den is y has e ol ed o
a p os he ically d i en implan placemen concep , meaning ha biology, biomechanics,
unc ion and es he ics o he implan suppo ed ehabili a ion should be conside ed o he
adequa e implan posi ion in bone. Al hough p ope amoun o bone is needed o go along
wi h he es he ical and unc ional p os he ic design, a iable disc epancies in he a ailable
bone a e seldom ound. This may occu because o p olonged oo h loss, auma, inju y o
bone disease and esec ion, conduc ing o a ho izon al, e ical o combined bone de ec
(Siebe ). Hence, se e al echniques and ma e ials o bone augmen a ion ha e been used
concomi an wi h implan placemen o as a s aged app oach [1–4].
Following he biological p inciples o selec i e cell exclusion o egene a i e wound
healing and guided issue egene a ion, hese we e la e p o en o be applicable o guided
bone egene a ion also. Techniques in ol e placing a mechanical ba ie o p o ec he
blood clo and o isola e he bony de ec om he su ounding connec i e and epi helial
issue in asion. This space is needed o allow he os eoblas s o access he space in ended
o bone egene a ion [5,6].
Ti anium igid sca olds we e success ully used o bone augmen a ion, e en ou side
o he bone en elope. P esen ly, one mains eam di ec ion o 3D p in ing is biomedical
applica ions, speci ically in c ea ing sca olds o medical implan s such as indi idualized
i anium meshes o bone egene a ion [
7
–
9
]. In ecen yea s, he de elopmen o pe sonal-
ized apid p o o yping medical de ices based on he digi al imaging and communica ions
in medicine (DICOM) iles p o ided by compu e ized omog aphy/cone beam compu -
e ized omog aphy (CT/CBCT) scans has deeply in ensi ied [
10
]. Based on he pa ien ’s
bone de ec and eso ing o compu e aided design (CAD) so wa e, i is possible o design
medical de ices wi h he in en o ec ea ing he los idimensional bone ana omy.
Rega dless o he p oduc ion echnique o any implan able de ices, i is manda o y
o con ol he cha ac e is ics such as pe meabili y, su ace opog aphy and oughness, and
op imize hei biological pe o mance [
11
–
16
]. High deg ees o oughness ep esen a majo
isk o ionic leakage om he ma e ial [
17
] and he bac e ial adhesion can be inc eased,
wi h he consequence o implan ailu es [
10
]. Smoo h su aces a e able o slow down
he biological p ocesses a he in e ace, keeping he i anium oxidized laye p ope ies
una ec ed o longe ime pe iods [
9
]. The associa ed co ec mic o- and nano- oughness
le el can s imula e os eoblas di e en ia ion, p oli e a ion and p oduc ion o bo h ma ix
and local g ow h ac o s [
10
]. Fu he mo e, changes in oughness co ela e wi h selec i e
p o ein adso p ion, collagen syn hesis and he ma u a ion o chond ocy es, which all
signi ican ly in luence he implan ’s osseoin eg a ion [10].
I is well known ha he implan –li ing issues in e ac ions depend on he su ace
p ope ies, such as oughness, we abili y, su ace ene gy and chemical composi ion, among
o he s. Bioma e ials esea ch should op imize, a di e en scales, he su ace cha ac e is ics
in o de o imp o e di e en unc ions: bioac i i y, osseoin eg a ion o bac e icide beha io .
In addi ion, i anium meshes a e suscep ible o co osion due o he p esence o me als
o di e en chemical na u e in he mou h, as well as he elease o i anium ions in o he
en i onmen which mus be aken in o accoun [
11
–
13
]. I has been long ecognized ha he
co osion p oduc s o med as a esul o me al–en i onmen in e ac ions ha e a signi ican
bea ing on he biocompa ibili y and long- e m s abili y o he p os heses/implan . The
ma e ial used mus no cause any biological ad e se eac ion and mus e ain i s o m
and p ope ies [
11
,
12
] du ing unc ion. Human s oma ogna hus is subjec ed o a ying
changes in pH and empe a u e owing o di e ences in local, sys emic, en i onmen al,
economic and social condi ions o each indi idual. Co osion can esul om he p esence
o a numbe o co osi e species such as hyd ogen ion (H
+
), sul ide compounds (S
2−
),
dissol ed oxygen, ee adicals (O
2−
, O
−
), and chlo ide ion (Cl
−
) esul ing in he me al
su ace b eakdown and a consequen ad e se issue eac ion [
13
]. In addi ion, he e ec o
Coa ings 2022,12, 154 3 o 17
bac e ia can lead o he appea ance o bac e ial plaque which will a ec bone egene a ion
and cause in lamma ion in he pa ien [14–16].
Passi a ion is, in gene al, an oxida ion eac ion ob ained by chemical o elec ochem-
ical p ocess which p omo es he o ma ion and inc easing o he hickness o p o ec i e
laye s [
10
–
13
]. The e ec o passi a ion and oxida i e agen s and he ole o i anium
oxide as he physico-chemical cha ac e is ics o he su ace a e poo ly s udied and unde -
s ood [17–20].
In i o
s udies ha e implied ha he nega i ely cha ged and hyd ophilic TiO
2
laye
is, in ac , he key ac o o he o e all biocompa ibili y as i egula es he p o ein ad-
so p ion [
9
]. Fo he pa icula case o he den is y, coun less s udies ha e al eady been
conduc ed in o de o gua an ee he implan a ion sa e y. Usually, no in lamma o y esponse
signs a e ound in he o al issue adjacen o i anium implan s; howe e , i is impo an o
no e ha o some pa ien s, hype sensi i i y can be induced [9].
In his wo k, he aim was o s udy an al e na i e passi a ion me hod using he so-
called Pi anha solu ion. The Pi anha solu ion is a mix u e o sul u ic acid and hyd ogen
pe oxide. We s udied he e ec s o Pi anha solu ion ea men on su ace physical-chemical
p ope ies, chemical deg ada ion (co osion and elease o ions) and an imic obial ac i i y
agains G am-posi i e and G am-nega i e bac e ia.
2. Ma e ials and Me hods
2.1. Samples
One hund ed wen y G ade 5 i anium alloy (Ti6Al4V) meshes (BoneEasy, A ada,
Po ugal) we e used. Figu e 1shows he mesh and i s applica ion as a memb ane wi h
calcium phospha e.
We wo ked wi h 3 g oups o samples:
Con ol: as- ecei ed ma e ial.
HCl passi a ion: The meshes we e imme sed in a solu ion o hyd ochlo ic acid (HCl)
20% ( ) o 40 s a oom empe a u e (HCl g oup). This is he gold-s anda d passi a ion
ea men o den al implan s and p os hesis.
Pi anha passi a ion: The meshes we e imme sed in a solu ion o Pi anha, which is a
mix u e o sul u ic acid 96% ( ) and a 50:50 a io o hyd ochlo ic acid (HCl) 20% ( ) and
hyd ogen pe oxide 30% ( ) o 2 h.
Pi anha solu ions a e a mix u e o concen a ed sul u ic acid wi h hyd ogen pe oxide,
usually in a a io o 3:1 o 7:1. They a e used o emo e ace amoun s o o ganic esidues,
such as pho o esis , om subs a es. The mixing p ocedu e is an exo he mic eac ion
ha can each empe a u es o 100
◦
C o highe . The eac ion o hyd ogen pe oxide on
concen a ed sul u ic acid p oduces highly ac i a ed and oxidizing pe oxymonosul u ic
acid (H
2
SO
5
), also called Ca o’s acid [
1
]. Howe e , he e a e many di e en mix u e a ios
ha a e commonly used, and all a e called Pi anha. The addi ion o NH
4
OH in o de o
accele a e he decomposi ion o H
2
O
2
o he addi ion o HCl, as in his esea ch, a o s
cleanness and inc eases he oxide s abiliza ion. Pi anha solu ion mus be p epa ed wi h
g ea ca e. I is highly co osi e and an ex emely powe ul oxidize . Su aces mus be
easonably clean and comple ely ee o o ganic sol en s om p e ious washing s eps
be o e coming in o con ac wi h he solu ion. Pi anha solu ion cleans by decomposing
o ganic con aminan s, and a la ge amoun o con aminan will cause iolen bubbling and
a elease o gas ha can cause an explosion [21].
A e ea men , all samples we e cleaned a sequence o 3 ul asonic ba hs (3 min each):
wo consecu i e wi h dis illed wa e , ollowed by one wi h e hanol.
Coa ings 2022,12, 154 4 o 17
Coa ings 2022, 12, x FOR PEER REVIEW 4 o 18
Figu e 1. G ade 5 i anium mesh used in his s udy.
2.2. Su ace Cha ac e iza ion
Roughness o all g oups was de e mined using an Olympus LEXT OLS3100 con ocal
mic oscope (Olympus, Tokyo, Japan). Th ee samples pe g oup we e es ed and 3 meas-
u emen s pe sample we e aken a ×1000 magni ica ion. The pa ame e s Ra and Rz we e
de e mined. Ra co esponds o he a i hme ic mean o he absolu e alues o he de ia-
ions o he p o iles o a gi en leng h o he sample. Rz co esponds o he sum o he
maximum peak heigh and he maximum alley dep h wi hin he sampling leng h. [21].
The wa e sessile d op echnique was used o he measu emen o he con ac angle,
θ, o med be ween he wa e d op and he su ace. The g ea e he con ac angle, he lowe
he we abili y and ice e sa. Fo angles less han 10°, he su ace is conside ed supe hy-
d ophilic, o angles be ween 10° and 90° su aces a e hyd ophilic and o angles g ea e
han 90°, su aces a e conside ed hyd ophobic. A d ople gene a ion sys em equipped
wi h a 500 μL Hamil on sy inge wi h mic ome ic displacemen con ol was used o con-
ol he olume (3 μL) and o deposi he d ople . The analysis was pe o med using a
gonyome e wi h d op p o ile image cap u e (Con ac Angle Sys em OCA15plus,
Da aPhysics, Filde s ad , Ge many) and analyzed wi h SCA20 so wa e (Da aPhysics,
Filde s ad , Ge many) [22,23].
To calcula e he su ace ee ene gy, he con ac angle was measu ed wi h wo di e -
en liquids, wa e and diiodome hane. The con ac angle measu emen s o diiodome hane
we e ob ained ollowing he same p ocedu e used o measu e wa e con ac angles [22].
The su ace ee ene gy and i s pola (γp) and dispe si e (γd) componen s we e hen cal-
cula ed using he Owens and Wend equa ion [17]:
))()((2)cos1( 2/1
p
S
p
L
2/1d
S
d
LL 
(1)
Su ace mo phology o he samples was analyzed wi h a ocused ion beam Zeiss
Neon40 FE-SEM (Ca l Zeiss NTS GmbH, Obe kochen, Ge many). Images o uncoa ed
samples we e aken a a wo king dis ance o 7 mm and an accele a ing ol age o 5 kV.
An EDS de ec o (INCA Pen aFETx3 sys em, Ox o d Ins umen s, Abingdon, UK) was
used o de ec sil e p esence on he su ace o he samples. This mic oscope has a esol -
ing powe o 3 nm and allows he obse a ion o he nano ex u es p oduced by he eac-
ion o he Pi anha solu ion wi h he Ti6Al4V alloy.
Figu e 1. G ade 5 i anium mesh used in his s udy.
2.2. Su ace Cha ac e iza ion
Roughness o all g oups was de e mined using an Olympus LEXT OLS3100 con ocal
mic oscope (Olympus, Tokyo, Japan). Th ee samples pe g oup we e es ed and 3 mea-
su emen s pe sample we e aken a
×
1000 magni ica ion. The pa ame e s Ra and Rz we e
de e mined. Ra co esponds o he a i hme ic mean o he absolu e alues o he de ia ions
o he p o iles o a gi en leng h o he sample. Rz co esponds o he sum o he maximum
peak heigh and he maximum alley dep h wi hin he sampling leng h [21].
The wa e sessile d op echnique was used o he measu emen o he con ac angle,
θ
, o med be ween he wa e d op and he su ace. The g ea e he con ac angle, he
lowe he we abili y and ice e sa. Fo angles less han 10
◦
, he su ace is conside ed
supe hyd ophilic, o angles be ween 10
◦
and 90
◦
su aces a e hyd ophilic and o angles
g ea e han 90
◦
, su aces a e conside ed hyd ophobic. A d ople gene a ion sys em
equipped wi h a 500
µ
L Hamil on sy inge wi h mic ome ic displacemen con ol was
used o con ol he olume (3
µ
L) and o deposi he d ople . The analysis was pe o med
using a gonyome e wi h d op p o ile image cap u e (Con ac Angle Sys em OCA15plus,
Da aPhysics, Filde s ad , Ge many) and analyzed wi h SCA20 so wa e (Da aPhysics,
Filde s ad , Ge many) [22,23].
To calcula e he su ace ee ene gy, he con ac angle was measu ed wi h wo di e en
liquids, wa e and diiodome hane. The con ac angle measu emen s o diiodome hane we e
ob ained ollowing he same p ocedu e used o measu e wa e con ac angles [
22
]. The
su ace ee ene gy and i s pola (
γp
) and dispe si e (
γd
) componen s we e hen calcula ed
using he Owens and Wend equa ion [17]:
γL·(1+cos θ) = 2·((γd
L·γd
S)1/2 + (γp
L·γp
S)1/2)(1)
Su ace mo phology o he samples was analyzed wi h a ocused ion beam Zeiss
Neon40 FE-SEM (Ca l Zeiss NTS GmbH, Obe kochen, Ge many). Images o uncoa ed
samples we e aken a a wo king dis ance o 7 mm and an accele a ing ol age o 5 kV. An
EDS de ec o (INCA Pen aFETx3 sys em, Ox o d Ins umen s, Abingdon, UK) was used o
de ec sil e p esence on he su ace o he samples. This mic oscope has a esol ing powe
o 3 nm and allows he obse a ion o he nano ex u es p oduced by he eac ion o he
Pi anha solu ion wi h he Ti6Al4V alloy.
2.3. Co osion Beha io
A o al o 60 samples, (n= 20) o each g oup o samples, we e used o he co osion
es s. The es a ea o each sample was 19.6 mm
2
. The elec oly e o all es s was Hank’s
solu ion (Table 1), which is a saline luid ha closely cap u es he ion composi ion o he
human se um en i onmen .
Coa ings 2022,12, 154 5 o 17
Table 1. Composi ion o Hank’s solu ion.
Chemical P oduc Composi ion (mM)
K2HPO40.44
KCl 5.4
CaCl21.3
Na2HPO40.25
NaCl 137
NaHCO34.2
MgSO41.0
C6H12O65.5
The elec ochemical cell used was a polyp opylene (PP) con aine wi h a capaci y
o 185 mL and a me hac yla e lid wi h 6 holes o he in oduc ion o he sample, he
e e ence elec ode and he coun e elec ode (Figu e 2). Fo bo h he open ci cui po en ial
measu emen es s and he po en iodynamic es s, he e e ence elec ode used was a
calomel elec ode (sa u a ed KCl), wi h a po en ial o 0.241 V compa ed o he s anda d
hyd ogen elec ode. All es s we e pe o med a oom empe a u e and in a Fa aday cage
o a oid he in e ac ion o ex e nal elec ic ields.
Fo he open ci cui po en ial measu emen es s, only he sample and he e e ence
elec ode we e placed in he elec ochemical cell. Tes s we e ca ied ou o 5 h o all he
samples, aking measu emen s e e y 10 s. The po en ial was conside ed o be s abilized
when he a ia ion o he po en ial is less han 2 mV o e a pe iod o 30 min as indica ed
in he ASTM G31 s anda d [23]. This es assesses which ma e ials a e mo e noble (highe
po en ial) and hus, less suscep ible o co ode. The da a and he E- cu es we e ob ained
using he Powe Sui e so wa e (Schneiede Elec ic, Ruil-Malmaison, F ance) wi h he
Powe Co -Open ci cui (Schneiede Elec ic, Ruil-Malmaison, F ance).
Coa ings 2022, 12, x FOR PEER REVIEW 5 o 18
2.3. Co osion Beha io
A o al o 60 samples, (n = 20) o each g oup o samples, we e used o he co osion
es s. The es a ea o each sample was 19.6 mm2. The elec oly e o all es s was Hank’s
solu ion (Table 1), which is a saline luid ha closely cap u es he ion composi ion o he
human se um en i onmen .
Table 1. Composi ion o Hank’s solu ion.
Chemical P oduc
Composi ion (mM)
K2HPO4
0.44
KCl
5.4
CaCl2
1.3
Na2HPO4
0.25
NaCl
137
NaHCO3
4.2
MgSO4
1.0
C6H12O6
5.5
The elec ochemical cell used was a polyp opylene (PP) con aine wi h a capaci y o
185 mL and a me hac yla e lid wi h 6 holes o he in oduc ion o he sample, he e e -
ence elec ode and he coun e elec ode (Figu e 2). Fo bo h he open ci cui po en ial
measu emen es s and he po en iodynamic es s, he e e ence elec ode used was a cal-
omel elec ode (sa u a ed KCl), wi h a po en ial o 0.241 V compa ed o he s anda d hy-
d ogen elec ode. All es s we e pe o med a oom empe a u e and in a Fa aday cage o
a oid he in e ac ion o ex e nal elec ic ields.
Fo he open ci cui po en ial measu emen es s, only he sample and he e e ence
elec ode we e placed in he elec ochemical cell. Tes s we e ca ied ou o 5 h o all he
samples, aking measu emen s e e y 10 s. The po en ial was conside ed o be s abilized
when he a ia ion o he po en ial is less han 2 mV o e a pe iod o 30 min as indica ed
in he ASTM G31 s anda d [23]. This es assesses which ma e ials a e mo e noble (highe
po en ial) and hus, less suscep ible o co ode. The da a and he E- cu es we e ob ained
using he Powe Sui e so wa e (Schneiede Elec ic, Ruil-Malmaison, F ance) wi h he
Powe Co -Open ci cui (Schneiede Elec ic, Ruil-Malmaison, F ance).
Figu e 2. Expe imen al se up used o assessing co osion esis ance.
Cyclic po en iodynamic pola iza ion cu es we e ob ained o he 3 s udy g oups
ollowing he ASTM G5 s anda d. In his es , a a iable elec ical po en ial is imposed by
he po en ios a be ween he sample and he e e ence elec ode, causing a cu en o low
be ween he sample and he coun e elec ode. The coun e elec ode used was pla inum
[17,24–25]. Be o e s a ing he es , he sys em was allowed o s abilize by means o an
Figu e 2. Expe imen al se up used o assessing co osion esis ance.
Cyclic po en iodynamic pola iza ion cu es we e ob ained o he 3 s udy g oups
ollowing he ASTM G5 s anda d. In his es , a a iable elec ical po en ial is imposed
by he po en ios a be ween he sample and he e e ence elec ode, causing a cu en
o low be ween he sample and he coun e elec ode. The coun e elec ode used was
pla inum [17,24,25]. Be o e s a ing he es , he sys em was allowed o s abilize by means
o an open ci cui es o 1 h. A e s abiliza ion, he po en iodynamic es was launched,
pe o ming a cyclic sweep om
−
0.8 mV o 1.7 mV a a speed o 2 mV/s. These pa ame-
e s we e en e ed in o he Powe Sui e p og am using he Powe Co -Cyclic Pola iza ion
unc ion o ob ain he cu es. The pa ame e s s udied we e:
1. ico (µA/cm2)—co osion cu en densi y;
2.
E
co
(mV)—co osion po en ial: alue a which he cu en densi y changes om
ca hodic o anodic;
3. E ep (mV)— epassi a ion po en ial: po en ial a which he passi e laye egene a es;
4. Ep(mV)—pi ing po en ial: alue a which pi ing co osion may occu ;

Coa ings 2022,12, 154 6 o 17
5. ip(µA/cm2)—passi a ion cu en densi y;
6. ip (µA/cm2)— epassi a ion cu en densi y.
The E
co
and i
co
pa ame e s a e ob ained by ex apola ing he Ta el slopes. The
Ta el slopes a e also used o ob ain he Ta el coe icien s: anodic (
β
a) and ca hodic (
β
c).
These coe icien s ep esen he slopes o he anodic and ca hodic b anch, espec i ely. In
acco dance wi h he ASTM G102-89 s anda d [
23
–
26
], hese alues a e hen used o calcula e
he pola iza ion esis ance (R
p
) using he S e n–Gea y exp ession and he co osion a e
(CR in mm/yea ) [24–28].
Rp =βa·βc
2.303 ·(βa+βc)·ico (2)
The pola iza ion esis ance indica es he esis ance o he sample o co osion when
subjec ed o small a ia ions in po en ial. A o al o 30 po en iodynamic es s we e ca ied
ou , ob aining a leas 10 cu es pe g oup.
CR =K1·ico
ρ·EW (3)
2.4. Ion Release
Fi e samples om each g oup we e used o he me al ion eco e y es . A e weigh-
ing he samples (m = 0.206 g) and ollowing he ISO 10993-12 s anda d [
26
], a weigh
adjus men was made a he a e o 1 mL o Hank’s solu ion o each 0.2 g o sample, as
indica ed in he s anda d. The 5 samples o each g oup we e placed in he same Eppendo
wi h 5 mL o Hank’s solu ion and s o ed a 37
◦
C. Hank’s solu ion should be ex ac ed
and s o ed in he e ige a o a e 1, 3, 7, 14 and 21 days. A e each ex ac ion, 5 mL o
esh Hank’s solu ion has been eplenished in o he Eppendo con aining he samples. All
Eppendo ubes should be cleaned wi h 2% Ni ic Acid and d ied be o e use.
A e 21 days, he concen a ion o eleased i anium ions was measu ed, a he es
imes indica ed abo e, by induc i ely coupled plasma mass spec ome y (ICP-MS) wi h
he Agilen Technologies 7800 ICP-MS.
2.5. Bac e ia Analysis
Two ypes o bac e ia, P. ae uginosa (Colección española de cul i os ipo, CECT 110,
Valencia, Spain) and S. sanguinis (Cul u e Collec ion Uni e si y o Go henbu g, CCUG
15915, Go henbu g, Sweden), a G am-nega i e and a G am-posi i e s ain, espec i ely,
we e used o he bac e ial adhesion es . Th ee samples pe g oup and bac e ial s ain
we e es ed.
The cul u e media and ma e ial (PBS) we e p e iously s e ilized by au ocla ing a
121 ◦C
o 30 min. P io o he adhesion es , he samples we e also s e ilized. Fo his pu -
pose, h ee 5 min e hanol washes we e ca ied ou in s e ile cul u e pla es. A e emo ing
he e hanol, he samples we e exposed o ul a iole ligh o ano he 30 min [29,30].
The aga pla es we e cul u ed a 37
◦
C o 24 h. F om his cul u e, he liquid inoculum
was p epa ed by suspending he bac e ia in 5 mL o BHI (B ain Hea In usion) and
incuba ed o 24 h a 37
◦
C. The medium was hen dilu ed o an op ical densi y o 0.1
a a wa eleng h o 600 nm (OD600 = 0.1). Fo bac e ial adhesion, enough solu ion wi h
a concen a ion equi alen o OD600 = 0.1 o co e he su aces (500
µ
L/sample) was
in oduced in o he well o he cul u e pla e o each sample and incuba ed a 37
◦
C o 1 h.
A e his ime, he samples we e insed wi h PBS o 5 min wice and he bac e ia
we e ixed wi h a 2.5% glu a aldehyde solu ion in PBS (30 min in he e ige a o ). The
glu a aldehyde solu ion was hen emo ed and he samples we e insed wi h PBS 3 imes
o 5 min. Fo iabili y analysis by con ocal mic oscopy, he LIVE/DEAD BacLigh bac e ial
iabili y ki (The mo Fishe , Mad id, Spain) was used [
13
,
14
]. A solu ion was p epa ed
wi h 1.5
µ
L o p opidium in 1 mL o PBS. Using a mic opipe e, a d op o his solu ion
(app oxima ely 50
µ
L/sample) was deposi ed on he s udy su ace and a e incuba ion
a oom empe a u e in he da k o 15 min, he samples we e insed 3 imes wi h PBS o
Coa ings 2022,12, 154 7 o 17
5 min. The su aces we e hen obse ed unde a con ocal mic oscope. Th ee images pe
sample we e aken a 630
×
magni ica ion (
×
63 objec i e). Wa eleng hs o 488 and 561 nm
we e used o de ec bac e ia wi h non-comp omised memb anes (LIVE) and comp omised
memb anes (DEAD), espec i ely.
P io o he obse a ion o he samples by scanning elec on mic oscopy (SEM), he
samples we e dehyd a ed. Fo he dehyd a ion p ocess and he c i ical poin d ying,
10 min
washes we e ca ied ou wi h e hanol solu ions o g adual concen a ions o 30%, 50%, 70%,
80%, 90%, 95% and 100%. They we e hen le o d y o 24 h a oom empe a u e. Then,
samples we e coa ed wi h pla inum o 5 s be o e obse a ion unde he mic oscope. Ten
images o each sample we e aken a 20,000
×
magni ica ions o bac e ial quan i ica ion on
each su ace.
2.6. S a is ical Analysis
All esul s we e exp essed as mean and s anda d de ia ion excep o he bac e ial
adhesion es esul s which we e exp essed as median and s anda d e o . The compa a i e
T.TEST (wi h he Excel so wa e) was ca ied ou be ween he di e en g oups a 95%,
which means ha o alues o p< 0.05, he e a e signi ican di e ences.
3. Resul s
Figu e 3shows SEM images o he su aces o he i anium alloy a e passi a ion
ea men s. No signi ican a ia ions be ween he con ol and HCl ea men we e de ec ed
and bo h ypes o su aces clea ly showed machining ma ks. Machining ma ks in HCl-
passi a ed su aces we e ligh e han in as-machined su aces, p obably due o he e ec o
he highe concen a ion o he acid. Howe e , on he su ace o he samples subjec ed o he
Pi anha passi a ion ea men , he acid a ack almos comple ely emo ed he machining
ma ks and, no ably, p oduced a homogenously-dis ibu ed and commonly-ob ained su ace
nano ex u e in he o m o nanoca i ies (Figu e 4) [15].
Coa ings 2022, 12, x FOR PEER REVIEW 8 o 18
Figu e 3. (A) Su aces o g ade 5 Ti alloy ea ed wi h di e en passi a ion me hods; (B) a highe magni ica ions.
Figu e 4. Nano ex u e o i anium alloy a e Pi anha passi a ion ea men obse ed by high- eso-
lu ion scanning elec on mic oscopy.
The di e en passi a ion ea men s on he i anium alloy meshes, ei he wi h HCl
o Pi anha solu ion, did no al e he a e age oughness (Ra), as no s a is ically signi ican
di e ences we e obse ed wi h espec o he con ol g oup (Table 2). Howe e , he Pi a-
nha ea men showed s a is ically signi ican lowe Rz alues wi h espec o he o he
g oups. These esul s sugges ha he Pi anha solu ion ea men a acked he i anium,
educing machining ailu es and c ea ing an oxide laye ha educes he di e ences be-
ween alleys and peaks. The la ge di e ence be ween he Ra and Rz alues shows ha
we ha e wo ypes o ex u e (Figu e 5), one associa ed wi h he u ning ma ks esponsible
o he high Rz alues and he o he he nano ex u e associa ed wi h he passi a ion ea -
men .
Figu e 3.
(
A
) Su aces o g ade 5 Ti alloy ea ed wi h di e en passi a ion me hods; (
B
) a highe
magni ica ions.
Coa ings 2022,12, 154 8 o 17
Coa ings 2022, 12, x FOR PEER REVIEW 8 o 18
Figu e 3. (A) Su aces o g ade 5 Ti alloy ea ed wi h di e en passi a ion me hods; (B) a highe magni ica ions.
Figu e 4. Nano ex u e o i anium alloy a e Pi anha passi a ion ea men obse ed by high- eso-
lu ion scanning elec on mic oscopy.
The di e en passi a ion ea men s on he i anium alloy meshes, ei he wi h HCl
o Pi anha solu ion, did no al e he a e age oughness (Ra), as no s a is ically signi ican
di e ences we e obse ed wi h espec o he con ol g oup (Table 2). Howe e , he Pi a-
nha ea men showed s a is ically signi ican lowe Rz alues wi h espec o he o he
g oups. These esul s sugges ha he Pi anha solu ion ea men a acked he i anium,
educing machining ailu es and c ea ing an oxide laye ha educes he di e ences be-
ween alleys and peaks. The la ge di e ence be ween he Ra and Rz alues shows ha
we ha e wo ypes o ex u e (Figu e 5), one associa ed wi h he u ning ma ks esponsible
o he high Rz alues and he o he he nano ex u e associa ed wi h he passi a ion ea -
men .
Figu e 4.
Nano ex u e o i anium alloy a e Pi anha passi a ion ea men obse ed by high-
esolu ion scanning elec on mic oscopy.
The di e en passi a ion ea men s on he i anium alloy meshes, ei he wi h HCl o
Pi anha solu ion, did no al e he a e age oughness (Ra), as no s a is ically signi ican
di e ences we e obse ed wi h espec o he con ol g oup (Table 2). Howe e , he Pi anha
ea men showed s a is ically signi ican lowe R
z
alues wi h espec o he o he g oups.
These esul s sugges ha he Pi anha solu ion ea men a acked he i anium, educing
machining ailu es and c ea ing an oxide laye ha educes he di e ences be ween alleys
and peaks. The la ge di e ence be ween he R
a
and R
z
alues shows ha we ha e wo
ypes o ex u e (Figu e 5), one associa ed wi h he u ning ma ks esponsible o he high
Rz alues and he o he he nano ex u e associa ed wi h he passi a ion ea men .
Table 2.
Roughness alues, R
a
and R
z,
o i anium alloy su aces wi h di e en passi a ion ea -
men s. Di e en le e s in he same column deno e s a is ically signi ican di e ences (p< 0.05)
be ween g oups.
Mesh Ra
(µm)
Rz
(µm)
Con ol 0.12 ±0.03 (a) 4.95 ±0.76 (A)
HCl 0.14 ±0.08 (a) 4.87 ±0.90 (A)
Pi anha 0.12 ±0.05 (a) 1.90 ±0.73 (B)
Coa ings 2022, 12, x FOR PEER REVIEW 9 o 18
Table 2. Roughness alues, Ra and Rz, o i anium alloy su aces wi h di e en passi a ion ea -
men s. Di e en le e s in he same column deno e s a is ically signi ican di e ences (p < 0.05) be-
ween g oups.
Mesh
Ra
(μm)
Rz
(μm)
Con ol
0.12 ± 0.03 (a)
4.95 ± 0.76 (A)
HCl
0.14 ± 0.08 (a)
4.87 ± 0.90 (A)
Pi anha
0.12 ± 0.05 (a)
1.90 ± 0.73 (B)
Figu e 5. Roughness pa ame e s quan i ied wi h di e en passi a ion condi ions: (a) Ra and (b)
Rz.
We abili y, i.e., hyd ophilic/hyd ophobic cha ac e o he es ed su aces, was de e -
mined measu ing he wa e con ac angle wi h he sessile d op echnique (Table 3). Fi s ly,
as- ecei ed con ol su aces we e hyd ophobic wi h a con ac angle highe han 90°. Sec-
ondly, all passi a ed su aces had signi ican ly highe hyd ophilici y han un ea ed con-
ol su aces. Thi dly, he su aces passi a ed wi h Pi anha solu ion p oduced a signi i-
can ly highe hyd ophilic ma e ial han he su aces ea ed wi h HCl. Wa e con ac an-
gle, as well as pola and dispe si e componen s o SFE, a e plo ed in Figu e 6.
Co esponding wi h he esul s o he we abili y o he di e en su aces, he pola
componen o he su ace ee ene gy in he i anium alloy passi a ed wi h Pi anha solu-
ion was he highes among all es ed su aces. The di e ences in he dispe si e and pola
componen s o he su ace ee ene gy o all es ed su aces we e s a is ically signi ican
[31–35].
I is widely accep ed ha inc easing he pola componen o a ma e ial’s su ace en-
e gy p omo es ini ial adhesion and cell p oli e a ion [17].
Table 3. Con ac angles and componen s o he su ace ee ene gy o he di e en ly passi a ed
meshes.
Mesh
Θ Wa e
(°)
Θ Diidome hane
(°)
γd
(mJ/m2)
γp
(mJ/m2)
SFE
(mJ/m2)
Con ol
102.76 ± 7.00
48.40 ± 2.32
35.15 ± 1.28
0.12 ± 0.10
35.28 ± 1.35
HCl
86.37 ± 4.12
53.54 ± 0.92
32.39 ± 0.52
3.31 ± 1.28
35.70 ± 1.60
Pi anha
49.05 ± 7.67
34.12 ± 3.94
42.37 ± 1.79
16.52 ± 4.22
58.90 ± 4.11
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Con ol HCl Pi anha
Ra (μm)
a)
0
1
2
3
4
5
6
7
Con ol HCl Pi anha
Rz (μm)
b)
Figu e 5.
Roughness pa ame e s quan i ied wi h di e en passi a ion condi ions: (
a
) Ra and (
b
) Rz.
Coa ings 2022,12, 154 9 o 17
We abili y, i.e., hyd ophilic/hyd ophobic cha ac e o he es ed su aces, was de e -
mined measu ing he wa e con ac angle wi h he sessile d op echnique (Table 3). Fi s ly,
as- ecei ed con ol su aces we e hyd ophobic wi h a con ac angle highe han 90
◦
. Sec-
ondly, all passi a ed su aces had signi ican ly highe hyd ophilici y han un ea ed con ol
su aces. Thi dly, he su aces passi a ed wi h Pi anha solu ion p oduced a signi ican ly
highe hyd ophilic ma e ial han he su aces ea ed wi h HCl. Wa e con ac angle, as
well as pola and dispe si e componen s o SFE, a e plo ed in Figu e 6.
Co esponding wi h he esul s o he we abili y o he di e en su aces, he po-
la componen o he su ace ee ene gy in he i anium alloy passi a ed wi h Pi anha
solu ion was he highes among all es ed su aces. The di e ences in he dispe si e
and pola componen s o he su ace ee ene gy o all es ed su aces we e s a is ically
signi ican [31–35].
I is widely accep ed ha inc easing he pola componen o a ma e ial’s su ace ene gy
p omo es ini ial adhesion and cell p oli e a ion [17].
Table 3.
Con ac angles and componen s o he su ace ee ene gy o he di e en ly passi a ed meshes.
Mesh ΘWa e
(◦)
ΘDiidome hane
(◦)
γd
(mJ/m2)
γp
(mJ/m2)
SFE
(mJ/m2)
Con ol 102.76 ±7.00 48.40 ±2.32 35.15 ±1.28 0.12 ±0.10 35.28 ±1.35
HCl 86.37 ±4.12 53.54 ±0.92 32.39 ±0.52 3.31 ±1.28 35.70 ±1.60
Pi anha 49.05 ±7.67 34.12 ±3.94 42.37 ±1.79 16.52 ±4.22 58.90 ±4.11
Coa ings 2022, 12, x FOR PEER REVIEW 10 o 18
Figu e 6. θ alues (a) and SFE alues (b) o cpTi ea ed wi h di e en passi a ion condi ions.
Table 4 shows ha he highes open ci cui co osion po en ial alues (EOCP) we e
ob ained o i anium alloy su aces ea ed wi h HCl. The e o e, HCl passi a ion p o-
duces he su aces wi h he leas endency o co osion, and he e o e he bes co osion
beha io . Con e sely, su aces ea ed wi h he Pi ahna solu ion showed he lowes al-
ues in open ci cui , which indica ed he highes endency o co osion. The po en iody-
namic s udies con i med ha he ea men ha p oduced su aces wi h he bes co osion
esis ance was using HCl, as hese passi a ed su aces showed he lowes alues o co o-
sion cu en densi y (ico ) and co osion a e (Vc). In addi ion, he HCl- ea ed samples
show he highes esis ance o pola iza ion (Rp). The Pi anha solu ion should p oduce he
hickes p o ec i e TiO2 laye ; howe e , su aces passi a ed wi h Pi anha did no ha e an
imp o ed co osion beha io wi h espec o he con ol samples. Mo eo e , only in sam-
ples ea ed wi h Pi anha solu ion pi ing co osion could be obse ed a e he po en-
iodynamic es s (Figu e 7).
Table 4. Elec ochemical and co osion pa ame e s assessed o Ti alloy meshes wi h di e en pas-
si a ion ea men s.
Mesh
EOCP
(mV)
ico
(μA/cm2)
Rp
(MΩ/cm2)
Eco
(V)
Vc
(μm/yea )
Con ol
−196 ± 01
0.027 ± 0.008
2.428 ± 0.390
−361 ± 14
0.233 ± 0.066
HCl
−145 ± 11
0.018 ± 0.005
2.479 ± 0.083
−536 ± 39
0.176 ± 0.048
Pi anha
−206 ± 27
0.056 ± 0.006
1.102 ± 0.149
−447 ± 26
0.488 ±0.047
0
20
40
60
80
100
120
Con ol HCl Pi anha
θ, (o)
a)
0
10
20
30
40
50
60
70
Con ol HCl Pi anha
SFE To al (𝒎𝑱/𝒎𝟐)
ϒsd
ϒsp
b)
Figu e 6. θ alues (a) and SFE alues (b) o cpTi ea ed wi h di e en passi a ion condi ions.
Table 4shows ha he highes open ci cui co osion po en ial alues (E
OCP
) we e
ob ained o i anium alloy su aces ea ed wi h HCl. The e o e, HCl passi a ion p oduces
he su aces wi h he leas endency o co osion, and he e o e he bes co osion beha io .
Con e sely, su aces ea ed wi h he Pi ahna solu ion showed he lowes alues in open
ci cui , which indica ed he highes endency o co osion. The po en iodynamic s udies
con i med ha he ea men ha p oduced su aces wi h he bes co osion esis ance was
using HCl, as hese passi a ed su aces showed he lowes alues o co osion cu en
densi y (i
co
) and co osion a e (V
c
). In addi ion, he HCl- ea ed samples show he
highes esis ance o pola iza ion (R
p
). The Pi anha solu ion should p oduce he hickes
p o ec i e TiO
2
laye ; howe e , su aces passi a ed wi h Pi anha did no ha e an imp o ed
co osion beha io wi h espec o he con ol samples. Mo eo e , only in samples ea ed
wi h Pi anha solu ion pi ing co osion could be obse ed a e he po en iodynamic es s
(Figu e 7).
Coa ings 2022,12, 154 16 o 17
19.
Va iola, F.; F ancis-Zalzal, S.; Leduc, A.; Ba beau, J.; Nanci, A. Oxida i e nanopa e ning o i anium gene a es mesopo ous
su aces wi h an imic obial p ope ies. In . J. Nanomed. 2014,9, 2319–2325. [C ossRe ]
20.
B une e, D.M.; Cheh oudi, B. The e ec s o he su ace opog aphy o mic omachined i anium subs a a on cell beha io
in i o
and in i o. J. Biomech. Eng. 1999,121, 49–57. [C ossRe ]
21.
Jones, C.W. Applica ions o Hyd ogen Pe oxide and De i a i es. In RSC Clean Technology, Monog aphs; Royal Socie y o Chemis y:
Camb idge, UK, 1999.
22.
Bagno, A.; Di Bello, C. Su ace ea men s and oughness p ope ies o Ti-based bioma e ials. J. Ma e . Sci. Ma e . Med.
2004
,15,
939–945. [C ossRe ] [PubMed]
23.
Liu, Y.; Zhao, Q. In luence o su ace ene gy o modi ied su aces on bac e ial adhesion. Biophys. Chem.
2005
,117, 39–46.
[C ossRe ] [PubMed]
24.
ASTM-E3-11; S anda d Guide o P epa a ion o Me allog aphic Specimens; ASTM In e na ional: Wes Conshohocken, PA,
USA, 2017.
25.
ASTM G5-14e1; S anda d Re e ence Tes Me hod o Making Po en ios a ic and Po en iodynamic Anodic Pola iza ion Measu e-
men s; ASTM In e na ional: Wes Conshohocken, PA, USA, 2014.
26.
ISO 10993-5:2009; Biological E alua ion o Medical De ices. Pa 5: Tes s o
In Vi o
Cy o oxici y; In e na ional O ganiza ion o
S anda diza ion: Gene e, Swi ze land, 2009.
27.
ASTM G-102-89; S anda d P ac ice o Calcula ion o Co osion Ra es and Rela ed In o ma ion om Elec ochemical Measu e-
men s; ASTM In e na ional: Wes Conshohocken, PA, USA, 2010.
28.
Gil, F.J.; Rod íguez, D.; Planell, J.A.; Co ada, M.; Gine , L.; Cos a, S. Gal anic co osion beha iou o Ti anium implan s coupled
o den al alloys. J. Ma . Sci. Ma . Med. 2000,11, 287–293.
29.
Gil, F.J.; Sánchez, L.A.; Espias, A.; Planell, J.A.
In i o
co osion beha iou and me allic ion elease o di e en p os hodon ic
alloys. In . Den . J. 1999,49, 347–351. [C ossRe ]
30. Al-Hi y, R.R.; Kappe , H.F.; Vienno , S.; Dala d, F.; G osgogea , B. Co osion esis ance measu emen s o den al alloys, a e hey
co ela ed? Den . Ma e . 2007,23, 679–687. [C ossRe ] [PubMed]
31.
Soc ansky, S.S.; Ha ajee, A.D.; Cugini, M.A.; Smi h, C.; Ken , R.L. Mic obial complexes in subgingi al plaque. J. Clin. Pe iodon ol.
1998,25, 134–144. [C ossRe ]
32.
Godoy-Galla do, M.; Wang, Z.; Shen, Y.; Mane o, J.M.; Gil, F.J.; Rod iguez, D.; Haapasalo, M. An ibac e ial coa ings on i anium
su aces: A compa ison s udy be ween
in i o
single-species and mul ispecies bio ilm. ACS Appl. Ma e . In e aces
2015
,7,
599–601. [C ossRe ]
33.
Yi, J.-H.; Be na d, C.; Va iola, F.; Zalzal, S.F.; Wues , J.D.; Rosei, F.; Nanci, A. Cha ac e iza ion o a bioac i e nano ex u ed su ace
c ea ed by con olled chemical oxida ion o i anium. Su . Sci. 2006,600, 4613–4621. [C ossRe ]
34. Cas ne , D.G.; Ra ne , B.D. Biomedical su ace science: Founda ions o on ie s. Su . Sci. 2002,500, 28–60. [C ossRe ]
35.
Wheelis, S.E.; Gind i, I.M.; Valde ama, P.; Wilson, T.G., J .; Huang, J.; Rod igues, D.C. E ec s o decon amina ion solu ions on he
su ace o i anium: In es iga ion o su ace mo phology, composi ion, and oughness. Clin. O al Implan s Res.
2015
,27, 329–340.
[C ossRe ]
36.
Hei z-May ield, L.J.; Lang, N.P. Compa a i e biology o ch onic and agg essi e pe iodon i is s. pe i-implan i is. Pe iodon ol 2000
2010,531, 67. [C ossRe ] [PubMed]
37.
Michia di, A.; Apa icio, C.; Ra ne , B.D.; Planell, J.A.; Gil, J. The in luence o su ace ene gy on compe i i e p o ein adso p ion on
oxidized NiTi su aces. Bioma e ials 2007,28, 586–594. [C ossRe ] [PubMed]
38.
Va iola, F.; Yi, J.H.; Riche , L.; Wues , J.D.; Rosei, F.; Nanci, A. Tailo ing he su ace p ope ies o Ti6Al4V by con olled chemical
oxida ion. Bioma e ials 2008,29, 1285–1298. [C ossRe ]
39.
Muhonen, V.; Heikkinen, R.; Danilo , A.; Jamsa, T.; Tuukkanen, J. The e ec s o oxide hickness on os eoblas a achmen and
su i al on NiTi alloy. J. Ma e . Sci. Ma e . Med. 2007,18, 959–967. [C ossRe ] [PubMed]
40.
Amo , S.B.; Baud, G.; Besse, J.P.; Jacque , M. S uc u al and op ical p ope ies o spu e ed i ania ilms. Ma e . Sci. Eng. B
1997
,47,
110–118. [C ossRe ]
41.
Vel en, D.; Biehl, V.; Aube in, F.; Valeske, B.; Possa , W.; B eme, J. P epa a ion o TiO
2
laye s on cp-Ti and Ti
6
Al
4
V by he mal and
anodic oxida ion and by sol-gel coa ing echniques and hei cha ac e iza ion. J. Biomed. Ma e . Res. 2002,59, 18–28. [C ossRe ]
42.
Amo , S.B.; Gued i, L.; Baud, G.; Jacque , M.; Ghedi a, M. In luence o he empe a u e on he p ope ies o spu e ed i anium
oxide ilms. Ma e . Chem. Phys. 2002,77, 903–911. [C ossRe ]
43.
McCa e y, E.; Wigh man, J.P. An X- ay pho oelec on spec oscopy spu e p o ile s udy o he na i e ai - o med oxide ilm on
i anium. Appl. Su . Sci. 1999,143, 92–100. [C ossRe ]
44.
A ys, A.; Philippa , C.; Dou o , N.; He, Y.; Le, Q.T.; Pi eaux, J.J. Analysis o i anium den al implan s a e ailu e o osseoin eg a-
ion: Combined his ological, elec on mic oscopy, and X- ay pho oelec on spec oscopy app oach. J. Biomed. Ma e . Res.
1998
,43,
300–312. [C ossRe ]
45.
Lee, T.M.; Chang, E.; Yang, C.Y. Su ace cha ac e is ics o Ti6Al4V alloy: E ec o ma e ials, passi a ion and au ocla ing. J. Ma e .
Sci. Ma e . Med. 1998,9, 439–448. [C ossRe ]
46.
Pouilleau, J.; De illie s, D.; Ga ido, F.; Du and-Vidal, S.; Mahe, E. S uc u e and composi ion o passi e i anium oxide ilms.
Ma e . Sci. Eng. B 1997,47, 235–243. [C ossRe ]

Coa ings 2022,12, 154 17 o 17
47. Lisowski, W.; an den Be g, A.H.J.; Smi he s, M. Cha ac e iza ion o i anium hyd ide ilm a e long- e m ai in e ac ion: SEM,
ARXPS and AES dep h p o ile s udies. Su . In e ace Anal. 1998,26, 213–219. [C ossRe ]
48.
Pegue oles, M.; Apa icio, C.; Bosio, M.; Engel, E.; Gil, F.J.; Planell, J.A.; Al anko , G. Spa ial o ganiza ion o os eoblas ib onec in
ma ix on i anium su aces: E ec s o oughness, chemical he e ogenei y and su ace ene gy. Ac a Bioma e .
2010
,6, 291–301.
[C ossRe ] [PubMed]
49.
Mukaddam, K.; As aso -F auenho e , M.; Fasle -Kan, E.; Ma o , L.; Kisiel, M.; Meye , E.; Köse , J.; Wase , M.; Bo ns ein, M.M.;
Kühl, S. E ec o a Nanos uc u ed Ti anium Su ace on Gingi al Cell Adhesion, Viabili y and P ope ies agains P. gingi alis.
Ma e ials 2021,14, 7686. [C ossRe ] [PubMed]
50.
I ano a, E.P.; Hasan, J.; Webb, H.K.; T uong, V.K.; Wa son, G.S.; Wa son, J.A.; Baulin, V.A.; Pogodin, S.; Wang, J.Y.;
Tobin, M.J.; e al.
Na u al Bac e icidal Su aces: Mechanical Rup u e o Pseudomonas Ae uginosa Cells by Cicada Wings. Small
2012
,8, 2489–2494.
[C ossRe ] [PubMed]
51.
I ano a, E.P.; Hasan, J.; Webb, H.K.; Ge inskas, G.; Juodkazis, S.; T uong, V.K.; Wu, A.H.F.; Lamb, R.N.; Baulin, V.A.; Wa son,
G.S.; e al. Bac e icidal Ac i i y o Black Silicon. Na . Commun. 2013,4, 2838. [C ossRe ] [PubMed]
52.
Se ano, C.; Ga cía-Fe nández, L.; Fe nández-Blázquez, J.P.; Ba beck, M.; Ghanaa i, S.; Unge , R.; Ki kpa ick, J.; A z , E.; Funk, L.;
Tu ón, P.; e al. Nanos uc u ed Medical Su u es wi h An ibac e ial P ope ies. Bioma e ials 2015,52, 291–300. [C ossRe ]
53.
Jenkins, J.; Man ell, J.; Neal, C.; Gholinia, A.; Ve kade, P.; Nobbs, A.H.; Su, B. An ibac e ial E ec s o Nanopilla Su aces A e
Media ed by Cell Impedance, Pene a ion and Induc ion o Oxida i e S ess. Na . Commun. 2020,11, 1626. [C ossRe ]
54.
Xu, Z.; He, Y.; Zeng, X.; Zeng, X.; Huang, J.; Lin, X.; Chen, J. Enhanced Human Gingi al Fib oblas Response and Reduced
Po phy omonas Gingi alis Adhesion wi h Ti ania Nano ubes. Biomed. Res. In . 2020,2020, 5651780. [C ossRe ]
55.
Ki an, A.S.; Kuma , T.S.; Pe umal, G.; Sangha i, R.; Doble, M.; Ramak ishna, S. Dual nano ib ous bioac i e coa ing and
an imic obial su ace ea men o in ec ion esis an i anium implan s. P og ess in O ganic Coa ings
2018
,121, 112–119. [C ossRe ]
56.
Seddiki, O.; Ha nagea, C.; Le esque, L.; Man o ani, D.; Rosei, F. E idence o an ibac e ial ac i i y on i aniumsu aces h ough
nano ex u es. Appl. Su . Sci. 2014,308, 275–284. [C ossRe ]
57.
Skinde soe, M.E.; K og el , K.A.; Blom, A.; Jiang, G.; P es wich, G.D.; Mansell, J.P. Dual Ac ion o Lysophospha ida e-
Func ionalised Ti anium:In e ac ions wi h Human (MG63) Os eoblas s and Me hicillin Resis an S aphylococcus au eus. PLoS
ONE 2015,10, e0143509. [C ossRe ] [PubMed]
58.
Ve meulen, N.; We den, J.; Keele , W.J.; Nandakuma , K.; Leung, K.T. The Bac e icidal E ec o Ul a iole and Visible Ligh on
Esche ichia Coli. Bio echnol. Bioeng. 2008,99, 550–556. [C ossRe ] [PubMed]