Hyb id coa ings o o hopaedic implan s o med by physical apou
deposi ion and mic oa c oxida ion
Roman Gabo
a
, Ladisla C c
ˇek
b
, Ma ina Doubko á
c,d
, Václa Nehasil
e
, Jose Hlinka
,g
, Pe Unucka
h
,
Ma e
ˇj Bu
ˇil
b
, Adéla Podep
ˇelo á
g
, Jana Seidle o á
a
, Lucie Bac
ˇáko á
c
a
Nano echnology Cen e, CEET, VSB – Technical Uni e si y o Os a a, 17, lis opadu 15/2172, 708 00 Os a a-Po uba, Czech Republic
b
Depa men o Ma e ials Enginee ing, Facul y o Mechanical Enginee ing, Czech Technical Uni e si y in P ague, Ka lo o náme
ˇs í 293/13, 120 00 P ague 2, Czech Republic
c
Labo a o y o Bioma e ials and Tissue Enginee ing, Ins i u e o Physiology o he Czech Academy o Sciences, . .i., Videnska 1083, 142 20 P ague 4, Czech Republic
d
Cha les Uni e si y, Second Facul y o Medicine, V U alu 84, 150 06 P ague 5, Czech Republic
e
Depa men o Su ace and Plasma Science, Cha les Uni e si y, P ague, Czech Republic
Depa men o Ma e ials Enginee ing, Facul y o Ma e ials and Technology, VSB-Technical Uni e si y o Os a a, 17, lis opadu 2172/15, 708 00 Os a a-Po uba, Czech Republic
g
Cen e o Ad anced Inno a ion Technologies, VSB-Technical Uni e si y o Os a a, 17, lis opadu 2172/15, 708 00 Os a a-Po uba, Czech Republic
h
VUHZ, a.s., 739 51 Dob a, Czech Republic
highligh s
Unique combina ion o PVD and MAO
echniques was used o p epa e
hyb id coa ings o imp o e Ti-6Al-4V
alloy p ope ies.
P epa ed hyb id coa ings wi h Z Ti
exhibi ed imp o ed co osion and
ab asion esis ance, and a lowe
coe icien o ic ion.
Hyd ophilic oxide s uc u e o
p epa ed hyb id coa ings inc eased
os eogenic di e en ia ion o human
bone ma ow mesenchymal s em
cells in i o.
Combined use o MAO nad PVD
echniques p o ides new possibili ies
o ine uning he ma e ial su ace
laye p ope ies.
g aphical abs ac
a icle in o
A icle his o y:
Recei ed 14 Decembe 2021
Re ised 29 May 2022
Accep ed 31 May 2022
A ailable online 3 June 2022
Keywo ds:
Ti-6Al-4V
PVD coa ing
MAO p ocess
Co osion p o ec ion
T ibology
Mesenchymal s em cells
abs ac
This s udy is ocused on he p epa a ion o new hyb id laye s in ended o su ace modi ica ion o Ti-6Al-
4V alloys o po en ial o hopaedic and den al applica ions. Combina ion o he echnology o physical
apou deposi ion (PVD) and subsequen mic o-a c oxida ion (MAO) was u ilized o he deposi ion o
Ti and Z Ti o o m hyb id oxide laye s. The oxide laye s we e p epa ed using an alkaline elec oly e wi h
glyce ol as an addi i e unde mic o-a c discha ge condi ions wi h di e en Si con en on hei su aces.
The hyb id Z Ti coa ings wi h a Z /Si s uc u e achie ed he bes ibological p ope ies desc ibed by a
low ic ion coe icien o 0.3 and high ab asion esis ance. The e was also an inc ease in co osion po en-
ial and pola iza ion esis ance o hyb id Z Ti coa ings. Al hough he p oli e a ion o human bone ma ow
mesenchymal s em cells was slowe on hese hyd ophilic Ti and Z Ti coa ings han bo h on uncoa ed Ti-
6Al-4V and he e e ence issue cul u e polys y ene dishes, bo h ypes o hyb id coa ing p omo ed
g ea e os eogenic di e en ia ion o hese cells, indica ed by app ox. wice as high ac i i y o alkaline
phospha ase. The hyb id oxide laye s newly de eloped in his s udy – especially he laye s wi h Z –
a e he e o e p omising o coa ing me allic bone implan s.
Ó2022 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
h ps://doi.o g/10.1016/j.ma des.2022.110811
0264-1275/Ó2022 The Au ho (s). Published by Else ie L d.
This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Ma e ials & Design 219 (2022) 110811
Con en s lis s a ailable a ScienceDi ec
Ma e ials & Design
jou nal homepage: www.else ie .com/loca e/ma des
1. In oduc ion
The design and he uses o bioma e ials o bone implan a ion
a e closely bound o hei s eng h, modulus o elas ici y, wea
esis ance and non- oxici y, osseoin eg a ion o bioine ness. High
osseoin eg a ion po en ial, i.e. a ac i eness o he adhesion,
g ow h and pheno ypic ma u a ion o bone cells, is needed o
long- e m/pe manen o hopedic implan s, such as s ems o
eplacemen s o la ge and small join s (e.g., hip, knee, shoulde ,
and apeziome aca pal join s) and o s oma ological implan s,
especially o pa s which a e in eg a ed in o he bone (e.g., he
s ems and cups o hip join eplacemen s). Fi m osseoin eg a ion
o hese implan s imp o es hei s abili y, p olongs hei li e ime
and, a he same ime, educes he likelihood o ailu e and he
need o eope a ion. Bioine ness, i.e. epulsi eness o a biocom-
pa ible ma e ial o cell adhesion and g ow h, is needed o
sho - e m/ empo a y implan s, e.g. o auma ological implan s
such as splin s, wi es, sc ews o spikes, so ha hey can be
emo ed easily a e he bone damage has healed [1].
The excellen co osi e and mechanical p ope ies o pu e Ti
and Ti alloys make hem sui able o applica ion in bo h ypes o
bone implan s men ioned abo e [2]. Signi ican ly be e mechani-
cal p ope ies we e achie ed ia s abilisa ion o he
a
+bphases in
Ti-6Al-4V alloy [3]. Ti anium alloys wi h a s abilised bphase, con-
aining elemen s such as Nb, Ta o Z , a e becoming mo e and mo e
popula . The main bene i s o hese alloys a e hei lowe modulus
o elas ici y and he absence o oxic elemen s such as Al and V [1].
G owing e idence o he elease o hese elemen s in o he body is
being ga he ed. This elease is linked wi h neu odegene a i e dis-
eases like Alzheime and Pa kinson, pe iphe al neu opa hy, and
also can a ec nega i ely he p oli e a ion o adjacen bone cells,
ex acellula ma ix syn hesis and he o ma ion o bone apa i e,
which an lead o os eomalacia [4]. The cy o oxici y o hese ele-
men s is u he po en ia ed o enhanced by he elease o o he
me al ion species (pa icula ly Ti), which is o inc easing impo -
ance, especially du ing iboco osion phenomena. This ype o
syne gis ic ad e se e ec o he eleased ions is well-known bo h
o pu e Ti and o i anium alloys, and i is he e o e c ucial o
add ess he ma e ial su ace deg ada ion p ocess in he human
body [5]. Ti anium i sel in he o m o ions, and in he o m o
nanopa icles and mic opa icles, can cause cy o oxic, in lamma-
o y, alle genic and e en mu agenic and ca cinogenic e ec s [6].
The well-known poo ibological p ope ies o Ti and i anium
alloys, accompanied by a high ic ion coe icien and insu icien
ab asion esis ance, place inc eased emphasis on he subsequen
su ace ea men ensu ing su icien ibological, co osion, and
biocompa ible p ope ies [7]. Comme cial coa ings o implan s
ha a e mos commonly applied o imp o e hese p ope ies
include diamond-like ca bon (DLC) [8], ni ide [9] and oxide ce a-
mic coa ings [10]. O he p omising bioac i e coa ings include a -
ious ino ganic subs ances, such as calcium phospha es o bioac i e
glass, and also o ganic subs ances, such as na u al polyme s (colla-
gen, gela in, cellulose, chi osan, algina e, hyalu onic acid) and syn-
he ic polyme s (polycap olac one, polye he e he ke one, poly-L-
lac ic acid, poly lac ic-co-glycolic acid, polyu e hane, poly inyl
alcohol), which can se e as ca ie ma ices o he deli e y o
he apeu ic d ugs, an imic obial agen s, g ow h ac o s and o he
biologically ac i e compounds [11].
S eady p og ess in he applica ion o p o ec i e coa ings in he
las wo decades has achie ed equi ed p ope ies such as wea
esis ance, good ibological p ope ies, oxida ion esis ance, co o-
sion esis ance, and a low coe icien o ic ion [12]. Coa ings p o-
iding hese p ope ies can be applied using physical apou
deposi ion (PVD) echnology [13], o example ca hodic a c e apo-
a ion (CAE), di ec cu en (DC) o pulsed DC magne on spu e -
ing [14], adio equency (RF) magne on spu e ing [15], and high
powe impulse magne on spu e ing (HIPIMS), which is widely
applied oday [16]. O he impo an coa ing echnologies include
chemical apo deposi ion (CVD), elec opho e ic deposi ion, sol–
gel deposi ion, laye -by-laye deposi ion, biomime ic deposi ion,
dip coa ing, d op coa ing, plasma sp aying and 3D p in ing [11].
A p omising al e na i e o he me hods men ioned abo e,
mic oa c oxida ion (MAO), also e e ed o as he plasma elec-
oly ic oxida ion (PEO) echnique, can be used o p epa e con in-
uous ce amic oxide laye s wi h excellen ly high adhesion o he
subs a e (e.g. Ti, Al, Nb, Z , Ta, H ), and wi h high esis ance o
wea and co osion. In he p epa a ion o ha d ce amic laye s, a
pulsed BI- o UNI-pola mode is used o p oduce he discha ge
obse ed du ing plasma elec olysis in a liquid elec oly e [10].
The oxide laye p epa ed by he MAO echnique consis s o a po -
ous amo phous ou e laye and a compac c ys alline inne laye
[17]. The g ow h o he ce amic laye (up o 20
l
m) occu s a em-
pe a u es o 10
3
-10
4
K and a a p essu e o 10
2
-10
3
Pa a e exceed-
ing he b eakdown ol age while he o ma ion o plasma channels
wi h a mic o-a c discha ge is aking place [18]. The su ace p ope -
ies o hese ce amic laye s, including hei po osi y, oughness,
and chemical composi ion, de e mine whe he hei success ul
applica ion ange is o sho - e m implan s o o long- e m
implan s [19]. The p ope ies o ce amic coa ings de ined in his
way a e a ec ed no only by he applied ol age, he pulse size,
he pulse wid h and he p ocess ime bu also by he composi ion
o he elec oly e ha is applied. The ions (Ca, P, Si) om he elec-
oly e a e inco po a ed in o he coa ing and a ec i s unc ional
p ope ies, including co osion esis ance, ab asion esis ance,
and biocompa ibili y [20]. The oughness and he po ous s uc u e
o he ou e po ous laye o he MAO coa ing nega i ely a ec he
esul ing coe icien o ic ion [21]. Al hough MAO coa ings
achie e high co osion esis ance, bo h laye ailu e and educed
co osion esis ance can occu unde loading. In an e o o elim-
ina e hese sho comings, many au ho s ha e in es iga ed he pos-
sibili y o doping he MAO elec oly e using nano pa icles TiO
2
[22], MoS
2
[23], Z O
2
[24] o y ium [25] and he use o seconda y
laye deposi ion in he o m o DLC coa ing o g aphene coa ing
[26]. The Al/Ti-6Al-4V duplex sys em p epa ed by a combina ion
o MAO and magne on spu e ing was also es ed wi h he aim
o imp o e he ibological p ope ies o he MAO coa ings [27].
The op ions a ailable in he use o he hyb id sys em p epa a ion
echnique allow a mo e de ailed s udy o he PVD-deposi ed laye
and i s inal p ope ies, achie ed by subsequen MAO. Applica ion
o his p ocedu e makes i possible, using PVD wi h he equi ed
unc ional p ope ies, o en ich he MAO laye s wi h he oxides
o deposi ed me als ha a e o med. To inc ease he os eoin eg a-
ion p ope ies, po ous Ca/P laye s on Ti-6Al-4V and Ti-29Nb-xH
subs a es we e p epa ed using a combina ion o MAO/PVD ech-
niques [28]. Subsequen ly, he RF magne on spu e ing echnique
was used on he p epa ed MAO po ous laye o he deposi ion o
an Mn laye [29], and a Zn laye [30].
Se e al wo ks [31,32] ha e s udied i anium alloys wi h Nb, Z ,
Ta, which – a e MAO ea men – achie ed inc eased wea esis-
ance and excellen co osion p ope ies due o he p esence o
s able oxides (e.g. Ta
2
O
5
, Z TiO
4
).
Due o hei high co osion esis ance, e y good mechanical
p ope ies and excellen biocompa ibili y, zi conium and i s alloys
a e p omising bioma e ials o o hopaedic implan s [33].MAO
applica ions - p e iously used o i anium and i s alloys - can also
be used o he p epa a ion o oxide laye s based on zi conium
[34]. The zi conium oxide laye s hus p epa ed can inc ease co o-
sion esis ance and wea esis ance, and can educe he coe icien
R. Gabo , L. C c
ˇek, M. Doubko á e al. Ma e ials & Design 219 (2022) 110811
2
o ic ion [35], while elimina ing he elease o oxic Al and V ions
om he Ti-6Al-4V alloy subs a e.
As p o ed by ou ea lie s udy [36], MAO enables he p epa a-
ion o highly hyd ophilic and bioine su aces ha educe cell
adhesion and g ow h wi hou a ec ing he cell iabili y, and u -
he modi ica ion o he MAO-deposi ed laye s can modula e he
su ace we abili y and a ac i eness o cell colonisa ion in a con-
ollable manne .
Op ions o eadily modi ying he p ocess pa ame e s o he
o ma ion and composi ion o a ma e ial coa ing a e aluable o
ine uning he su ace p ope ies o modula e he desi ed cell
esponse. Fo his pu pose, we p opose he applica ion o a no el
combina ion o su ace ea men and deposi ion echniques o
magne on spu e ing and MAO. Al hough each o hese ech-
niques is commonly used o modi ying ma e ials, hey ha e
no ye been sys ema ically used and esea ched oge he . The
combined use o hese echniques could p o ide an in e es ing
oppo uni y o s udy hei possibili ies wi h ega d o hei ma e-
ial p ope ies and hei pe o mance in biological en i onmen s
ep esen ed by cul u es o os eogenic cells in i o. In his s udy,
a combined PVD and MAO echnology was used o he de elop-
men o Ti and Z Ti laye s on a Ti-6Al-4V subs a e. These laye s
we e hen cha ac e ized in e ms o hei physical, chemical and
ibological p ope ies and hei in e ac ion wi h human bone
ma ow mesenchymal s em cells in cul u es on hei su ace.
We ound ha he deposi ion o bo h ypes o coa ings, pa icu-
la ly Z Ti, imp o ed he esis ance o he samples agains ic ion,
wea and co osion, and imp o ed he cell os eogenic
di e en ia ion.
2. Ma e ials and me hods
2.1. Sample p epa a ion
Round samples o Ti-6Al-4V i anium alloy (15 mm diame e ,
2.6 mm hickness) we e subjec ed o mass inishing o uni y he
su ace using an HV 20 ib a o y inishing machine (OTEC, Ge -
many) o 8 h wi h he use o KF 10 plas ic bodies. Ti and Z Ti
PVD laye s we e deposi ed on mechanically ea ed samples o
Ti-6Al-4V alloy in he Hauze Flexicoa 850 (Hauze , Ne he lands)
PVD uni based on ca hodic a c e apo a ion. The a ge s used o
a c e apo a ion o Ti and Z Ti we e 63 mm in diame e , and he
p essu e in he wo king chambe was 10
-3
mba du ing he appli-
ca ion. The p ocess pa ame e s o he PVD uni used o deposi ion
o he Ti and Z Ti laye s a e gi en in Table 1.
Samples wi h PVD laye s (Ti-PVD, Z Ti-PVD) deposi ed on a Ti-
6Al-4V subs a e we e modi ied unde MAO condi ions using a
pulse sou ce a 93 Hz, 7 % du y cycle, ol age 500 V eached in
30 s, ol age 500 V o 15 min (Fig. 1). Samples wi h MAO coa ings
(Ti-MAO, Z Ti-MAO) we e p epa ed in an elec oly e con aining
15 g/L Na
2
SiO
3
9H
2
O; 8 g/L NaOH; 80 g/L glyce ol (conduc i i y
28.8 mS/cm, pH 12.5).
2.2. Su ace analysis o he coa ings
The su aces and c oss-sec ions o he coa ings we e s udied
using he JEOL JSM-7610F Plus (JEOL, Japan) Scanning Elec on
Mic oscope (SEM) equipped wi h an au oemission ca hode as he
elec on sou ce. The samples we e scanned in BSE mode (20 keV
accele a ing ol age) wi h de ec ion o he seconda y elec ons.
The chemical composi ion o he coa ing was de e mined using
an ene gy dispe si e X- ay spec ome e (EDX, Ox o d Ins u-
men s). The KSG 110 Calo es de ice (INOVAP, Ge many) was used
o de e mine he hickness o he PVD coa ings. The MAO coa ing
hickness was s udied by SEM om polished c oss-sec ions in back
sca e emission mode. An a omic o ce mic oscope (AFM, Li eS-
cope
TM
) was used in semi-con ac mode o measu e he opog a-
phy and he su ace oughness o he sample su aces. The
co ela i e analysis also p o ides da a om bo h AFM and SEM
and allows a 3D co ela i e p obe and elec on mic oscopy iew
(CPEM, Neno ision s. .o.). Su ace oughness R
a
measu emen s
( he a i hme ic a e age o he absolu e alues o he p o ile
heigh s), R
measu emen s ( he e ical dis ance be ween he high-
es and lowes poin s o he p o ile) and R
z
measu emen s ( he
maximum heigh o he p o ile) we e ca ied ou in con ac mode
by AFM (AFM Li eScope
TM
).
The chemical s a es and he composi ion o he p epa ed laye s
we e analysed using he me hod o pho oelec on spec oscopy -
XPS (X- ay pho oelec on spec oscopy), which was ca ied ou in
an ul a- acuum appa a us wi h a basic p essu e o 1.10
-7
Pa. Pho-
oelec ons we e emi ed using an X- ay ube wi h a double anode
(Al, Mg). The Al K
a
1,2
line wi h p ima y ene gy o 1486.6 eV was
used in he expe imen s. XPS spec a we e eco ded using an Omi-
c on EA125 hemisphe ical analyse .
2.3. T ibological and adhesion es s
The ic ion coe icien and he wea a e we e es ed using a
CSM THT pin-on-disc ibome e (CSM Ins umen s, Swi ze land).
The ic ion pai consis ed o he es ed plana sample and an
Al
2
O
3
ball 6 mm in diame e . The es s we e pe o med a oom
empe a u e in phospha e-bu e ed saline (PBS), which se ed as
a simula ion o he human body en i onmen . The solu ion was
p epa ed by dissol ing one PBS able (Sigma-Ald ich, USA) in
200 mL o dis illed wa e . The solu ion con ained 10 mM phos-
pha e bu e , 2.7 mM KCl and 137.0 mM NaCl wi h pH 7.4 a 25 °C.
Du ing he ibological es , a no mal load o 1 N, linea sliding
speed 50 mm s
1
and numbe o laps 5000 wi h a adius o 6 mm
we e used. Each measu emen was epea ed wice o each sample.
The coe icien o ic ion (
l
) was calcula ed om he a io o he
angen ial ic ion o ce and he no mal o ce. The wea o he su -
ace o he Al
2
O
3
ball and he wid h o he wea ack on he plana
sample we e analysed using an Olympus DSX1000 (Olympus Co -
po a ion, Japan) digi al mic oscope a e he es . The wea a e
was calcula ed om he ollowing equa ion (A cha d,1953):
k=V/Fs
whe e kis he wea a e, Vis he wea olume, Fis he no mal load,
and sis he sliding dis ance. The wea olume was ob ained by mul-
iplying he a ea o he wea ack c oss-sec ion and he ci cum e -
ence o he wea ack. The wea ack c oss-sec ion a ea was
calcula ed om he wea ack p o ile, which was analysed by a
Zygo NewView 7200 op ical p o ilome e (Zygo Co po a ion, USA).
The CSM Re e es Xp ess+ (CSM Ins umen s, Swi ze land)
sc a ch es e was used o adhesion e alu ion. The sc a ch es e
was equipped wi h a Rockwell diamond inden e ( ip adius
200
l
m). The sc a ch es load was se o inc ease linea ly om
Table 1
P ocess pa ame e s.
Pa ame e s Ti Z Ti
Ta ge 99.98 % Ti 85 % Z / 15 % Ti
Coa ing p essu e (mba ) 5.0 10
-3
8.0 10
-3
Tempe a u e (°C) 430 430
Gas A A
Gas Flow (sccm) 540 380
Deposi ion ime (min) 180 150
Cu en on a ge (A) 60 80
Vol age bias (V) 80 80
R. Gabo , L. C c
ˇek, M. Doubko á e al. Ma e ials & Design 219 (2022) 110811
3
1 N o 30 N along he 5 mm sc a ch pa h wi h linea speed 10 mm.
min
1
.
2.4. Co osion es s
As he co osion esis ance o i anium and i s alloys is signi i-
can , di ec exposu e co osion es s would no be sui able; a
po en iodynamic pola isa ion me hod was he e o e used o
de e mining he co osion pa ame e s o he es ed samples. All
elec ochemical es s we e pe o med using a Vol alab PGZ 100
po en ios a equipped wi h Vol amas e 10 so wa e. The pa ame-
e s o he es we e used acco ding o he ASTM F 2129 and ASTM
F 746 s anda d es me hods [37]. PTFE/PE co osion cells wi h a
lowe exposu e hole (0.5 cm
2
) and a olume o 20 mL we e used
o es ing. The samples we e es ed in an iso onic physiological
solu ion (0.9 w % NaCl in double-dis illed H
2
O) in o de o simula e
he en i onmen o a li ing issue. No gas bubbling was applied
du ing es ing, and he empe a u e was se o 25 °C. A h ee-
elec ode se up was used, consis ing o a sample connec ed as a
wo king elec ode, a sa u a ed Calomel elec ode (SCE, +241 mV
s SHE) se as a e e ence elec ode, and a high pu i y ca bon od
connec ed as an auxilia y elec ode.
A 60-minu e ime lag was applied a e illing he co osion cells
wi h he solu ion o s abilise pa ial co osion p ocesses. A e he
ime gap, an elec ochemical impedance spec oscopy (EIS)
me hod was used o de e mine he elec ochemical pa ame e s
o he es ed su ace laye s. Be o e s a ing he po en iodynamic
pola isa ion, he ini ial po en ial alue was se o 70 mV s. he
open ci cui po en ial (OCP) a e s abilisa ion o he co osion
equilib ium, wi h he pola isa ion a e se o 60 mV.min
1
. The
dependence be ween he cu en lowing h ough he sample and
he po en ial applied o he es sample was eco ded du ing he
measu emen . The po en ial was g adually applied o he es ed
sample, which inc eased o e ime wi h he alue o he pola isa-
ion a e.
2.5. Con ac angle
The angle be ween he es ed su ace and he physiological
solu ion was e alua ed by he sessile d op me hod. The su ace
con ac angle was measu ed by he SEE sys em, and he ee su -
ace ene gy was calcula ed by Ad ex Ins umen so wa e (Ad ex
Ins umen s, s. .o., Czech Republic). Two
l
L d ople s o high pu i y
wa e we e applied o he es ed su ace, and he con ac angle h
was de e mined by he angen o he d op p o ile a he poin o
con ac o he h ee phases (liquid, solid and gas) wi h he line o
he su ace o he sample.
2.6. Cell cul u e condi ions
Human mesenchymal s em cells de i ed om bone ma ow
(hMSCs; No. 7500, ScienCell Resea ch Labo a o ies, USA) we e used
in all expe imen s. P io o he expe imen s, he hMSCs we e cul-
u ed in 75 cm
2
issue cul u e lasks (TPP, Swi ze land) in 15 mL
o Mesenchymal S em Cell Medium (MSCM medium; No.7501,
ScienCell Resea ch Labo a o ies, USA) o each ca 70% con luence.
The Ti-6Al-4V samples (Ti-6Al-4V, Ti-MAO, Z Ti-MAO) we e disin-
ec ed wi h e hanol o 2 h, we e insed in dH
2
O, we e s e ilised in
an au ocla e, and we e hen inse ed in o issue cul u e polys y -
ene 24-well pla es (TPP, Swi ze land). The hMSCs we e seeded
on hese samples a a densi y o 9,000 iable cells pe well (ca
5,000 cells/cm
2
) in 1.5 mL o
a
-MEM medium (No. 11900-016,
Gibco, USA) supplemen ed wi h 10% o e al bo ine se um (FBS),
L-glu amine (2 mmol/L; No. A2916801, Gibco, USA) and gen am-
icin (40
l
g/mL). In addi ion o he samples, he cells we e also
seeded di ec ly in o he issue cul u e pla e wells (PS), o p o ide
in o ma ion abou he s anda d cell beha iou . The cells we e cul-
u ed a 37 °C in a humidi ied a mosphe e wi h 5% CO
2
o 14 days.
In o de o suppo os eogenic di e en ia ion o he cells, he cul-
u e medium was en iched wi h asco bic acid (50
l
mol/L;
No. 49752-10G, Sigma-Ald ich, USA), b-glyce ophospha e
(20 mmol/L; No. G9422, Sigma-Ald ich) and dexame hasone
(10 nmol/L; No. D2915, Sigma-Ald ich). The medium was eplaced
wice pe week.
2.7. Fluo escence s aining
Fluo escence s aining was used o isualising and analysing he
cell sp eading a ea and mo phology (day1), and o e alua ing he
numbe and he iabili y o he cells g owing on he samples (day
1, 3, 7 and 14). The samples we e insed wi h PBS, hen ixed wi h
4% pa a o maldehyde (pH 7.4, 15 min), and we e insed again
wice wi h PBS. The ixed samples we e ea ed wi h 1% bo ine
se um albumin in PBS con aining 0.1% T i on X-100 (20 min) and
wi h 1% Tween 20 in PBS (20 min) (bo h Sigma-Ald ich, USA). Sub-
sequen ly, he cell nuclei we e s ained wi h DAPI (100 ng/mL, blue
luo escen signal; No. 32670, Sigma-Ald ich), he cell memb ane
and cy oplasm we e s ained wi h Texas Red C
2
-maleimide
(20 ng/mL, ed luo escen signal; Molecula P obes, In i ogen,
USA), o he F-ac in cy oskele on was s ained wi h TRITC-
conjuga ed phalloidin (100 ng/mL, ed luo escen signal; Sigma-
Ald ich). All dyes we e dilu ed in PBS and we e added o he cells
o 1 h a oom empe a u e (p o ec ed om ligh ).
A pa allel se o samples o each ime in e al was insed wi h
PBS and was s ained wi h a Li e/Dead Viabili y/Cy o oxici y ki o
mammalian cells (No. L3224, The moFishe Scien i ic, USA) acco d-
ing o he manu ac u e ’s ins uc ions. Calcein AM s ains he li ing
cells in g een, whe eas e hidium homodime 1 s ains he dead cells
wi h damaged cell memb anes in ed. Addi ional s aining o he
cell nuclei wi h DAPI was pe o med on day 7 and 14 o easie cell
coun ing.
Mic opho og aphs (20 pe samples/well) o andomly chosen
ields we e aken wi h an Olympus IX51 epi luo escence mic o-
scope, equipped wi h a DP70 came a (bo h om Olympus Co p.,
Japan). The mic opho og aphs we e analysed in ImageJ FIJI so -
wa e (h ps://imagej.ne /Fiji; 51). The ini ial cell sp eading a eas
a e p esen ed in
l
m
2
. The cell iabili y was de e mined as he pe -
cen age o li ing cells on he sample. The cell popula ion densi ies
on he samples a e p esen ed as he cell numbe pe cm
2
on a log
scale o assess p oli e a ion. The da a om day 1 and day 3 we e
used o calcula ing he cell popula ion doubling imes, acco ding
o he ollowing equa ion:
Fig. 1. Schema ic diag am o he deposi ion and laye modi ica ion p ocess applied o he Ti-6Al-4V subs a e ma e ial.
R. Gabo , L. C c
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4
DT ¼log2 
0
logN
logN
0
The ime in e als a e ep esen ed by
0
(day 1) and (day 3),
and N
0
and N ep esen he numbe o cells in a pa icula ime
in e al.
2.8. Resazu in assay
A esazu in assay based on he ac i i y o mi ochond ial
enzymes was used o in es iga e he me abolic ac i i y o he
hBMSC cells. On days 1, 3, 7 and 14 o cul i a ion, he samples we e
ans e ed o esh 24-well cul u e pla es wi h a esh cul u e
medium wi hou phenol ed supplemen ed wi h 10% o FBS. The
samples we e hen incuba ed wi h esazu in (No. R7017, Sigma–
Ald ich, USA) dilu ed o a inal concen a ion o 40
l
mol/L in a
esh cul u e medium wi hou phenol ed (1 mL/well) a 37 °Cin
a humidi ied a mosphe e con aining 5% CO
2
, p o ec ed om ligh .
A e 4 h (day 1 and 3) o 2.5 h (day 7 and 14) o incuba ion, he
luo escence was measu ed (Ex/Em = 530/590 nm) by a Syne gy
HT Mul i-Mode Mic opla e eade (BioTek, USA) in iplica e ali-
quo s. A solu ion incuba ed in a well wi hou cells se ed as a
blank con ol. The esul s we e co ec ed o he backg ound con-
ol (solu ion wi hou cells) and we e hen ela i ised o he es i-
ma ed o al numbe o cells g owing on each sample.
2.9. ALP assay
A quan i a i e analysis o alkaline phospha ase (ALP) enzyma ic
ac i i y o assess ea ly os eogenic di e en ia ion o he cells on he
samples was pe o med using 1-S ep PNPP Subs a e Solu ion (p-
ni ophenyl phospha e; No. 37621, The moFishe Scien i ic) on
days 7 and 14 o cul u e. The samples we e insed wi h PBS h ee
imes. A e a o al o 250
l
L o PNPP had been added o each sam-
ple, he cul u e pla e was incuba ed o 20 min a oom empe a-
u e o he eac ion o de elop. The abso bance o he wa e -
soluble yellow-colou p oduc was measu ed a 405 nm using a
Syne gy HT Mul i-Mode Mic opla e eade (BioTek, USA), in dupli-
ca es o each well. The abso bance esul s we e co ec ed o a
blank con ol (PNPP solu ion wi hou cells), and we e hen ela-
i ised o he es ima ed o al numbe o cells g owing on each
sample.
2.10. S a is ical analysis
All biological expe imen s we e pe o med independen ly h ee
imes wi h samples in iplica e in each ime in e al (n = 3 o
each expe imen al g oup). S a is ical analyses and da a isualiza-
ion we e pe o med in G aphPad P ism 8.3.0 (G aphPad So wa e,
USA). One Way ANOVA wi h Tukey’s es o K uskal-Wallis ANOVA
wi h Dunn’s pos hoc es we e pe o med on he da a om he
biological expe imen s. The da a a e exp essed in summa y g aphs
as median wi h in e qua ile ange (IQR). The esul s we e consid-
e ed s a is ically signi ican a p < 0.05.
3. Resul s and discussion
3.1. Mo phology and chemical analysis
Fig. 2a-c p esen s SEM images o he su ace s uc u e o he
subs a e and o he PVD coa ing. The images con i m he homoge-
neous and compac s uc u e o he coa ings ega ding hei hick-
ness and oughness, which was also de e mined by a Calo es
ins umen (Table 2). The esul s o he CPEM analysis, p esen ed
in Fig. 2d- , e ealed mic oscopic de ec s on he su aces (such as
aces o mechanical ea men o d ople s). The occu ence o
hese de ec s ollowing PVD deposi ion can be educed by lowe ing
he a c a ge cu en [38].
The SEM and CPEM images in Fig. 3a-d show ha he su ace
mo phology o he MAO coa ing is a compac po ous s uc u e wi h
an a e age po e size o abou 1
l
m. The o ma ion o a mic o-a c
discha ge was eco ded a e eaching 500 V, and i was ollowed
by a dec ease in cu en associa ed wi h he g ow h o he oxide
laye . The p esence o commonly known s uc u al mic oscopic
de ec s o he MAO coa ing in ol ing c acks was no con i med
on he s udied su aces wi h he unique CPEM echnology. The
de elopmen o he coa ing is accompanied by he o ma ion o
so-called olcanic c a e s (Fig. 3) du ing mic o-discha ges, when
he eleased mel o he subs a e (PVD laye ) is deposi ed ia dis-
cha ge channels on he su ace. Well- isible changes in su ace
opog aphy imaged using CPEM echnology indica e h ough 3D
imaging he p esence o c a e s ha a ec he esul ing oughness
o he hyb id MAO laye s compa ed o he su ace oughness o
he deposi ed PVD laye s (Fig. 2).
The esul ing measu ed hickness and oughness pa ame e s o
he MAO coa ings a e gi en in Table 2. The laye hicknesses we e
de e mined om he c oss-sec ions o indi idual MAO coa ings on
which he dis ibu ion o he elemen s p esen in he laye was
e alua ed. Figs. 4 and 5 show he p esence o he ou e and inne
MAO laye s. Elemen mapping om c oss-sec ions o he Ti-MAO
sample (Fig. 4) and o he Z Ti-MAO sample (Fig. 5) con i med bo h
he p esence o elemen s om PVD deposi ion and he elemen s
inco po a ed om he solu ion.
The esul ing alues o he po osi y s uc u e o he MAO coa -
ings (Table 2) con i med he inc ease in oughness in all h ee
moni o ed pa ame e s (R
a
,R
z
,R
) o he su aces o he MAO coa -
ings in compa ison wi h he ba e subs a e and he PVD coa ings
(Ti-PVD, Z Ti-PVD). The esul ing su ace s uc u e is due o he
high empe a u e (10
5
-10
7
K/s) and he p essu e in he discha ge
o he channels du ing he MAO p ocess. The esul ing mel passes
h ough he discha ge channel and, a e lea ing he channel, i
solidi ies in he elec oly e en i onmen [18]. The g ow h o he
su ace oughness and o he oxide laye is mainly associa ed wi h
he p ocess condi ions ( ol age, cu en densi y, ime, empe a u e,
elec oly e composi ion) and he p esence o addi i es. The e ec
o glyce ol as an addi i e on he su ace oughness and on he
s uc u e o he MAO coa ing was desc ibed in de ail in an ea lie
s udy by Gabo e al.[39]. In an e o o educe he su ace ough-
ness and o imp o e he ibological and co osi e p ope ies o he
coa ing, glyce ol was used as an addi i e in his wo k.
The di e en alues o he achie ed hickness and oughness o
he MAO coa ing (Table 2) a e caused by he di e en de elopmen
o he spa k discha ge, while he dis ibu ion and he in ensi y o
he spa k discha ge di ec ly a ec bo h he esul ing su ace mo -
phology and i s phase composi ion [40]. The cou se o he MAO
p ocess is in luenced by he di e en mel ing empe a u es o Ti
(1660 °C), Z (1852 °C) and o Z Ti, which may lead o mo e di icul
MAO de elopmen o he Z Ti-PVD sample, subsequen ly a ec ing
he esul ing su ace mic os uc u e [41]. The highe oughness
pa ame e s o he Ti-MAO coa ing a e caused by he g ea e in en-
si y and he la ge amoun o mic odischa ges on he su ace o he
sample han on he Z Ti-MAO coa ing. High mic odischa ge em-
pe a u es lead o dissolu ion o he subs a es and o subsequen
eac ions wi h he elec oly e elemen s ha a e p esen , including
apid solidi ica ion and he o ma ion o oxides ha a ec he
esul ing su ace mo phology [42].
C oss-sec ions and elemen al mapping con i m he phe-
nomenon o MAO coa ings, he p esence o a compac inne laye
and a po ous ou e laye (Figs. 4 and 5). The ou e laye is highly
po ous, ough, amo phous and no co osion- esis an . The dense
inne laye , cha ac e is ic o he MAO p ocess, is co osion-
esis an , c ys alline and i s hickness inc eases wi h he p ocess-
R. Gabo , L. C c
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5

Fig. 3. Mo phology o MAO coa ings. SEM images o Ti-MAO (a) and Z Ti-MAO (b); AFM images o Ti-MAO (c) and Z Ti-MAO (d).
Fig. 2. Mo phology o PVD coa ings. SEM images o he Ti-6Al-4V subs a e (a), Ti-PVD (b) and Z Ti-PVD (c); AFM images o he Ti-6Al-4V subs a e (d), Ti-PVD (e) and Z Ti-
PVD ( ).
Table 2
Su ace oughness and hickness o samples.
Sample Roughness (
l
m) MAO-coa ing (
l
m) PVD-coa ing (
l
m)
R
a
R
z
R inne laye ou e laye laye
Subs a e 0.09 ± 0.02 0.31 ± 0.62 0.55 ± 0.10 – – –
Ti-PVD 0.19 ± 0.09 0.64 ± 0.30 1.23 ± 0.30 – – 3.4 ± 0.50
Z Ti-PVD 0.24 ± 0.11 0.84 ± 0.38 1.55 ± 0.69 – – 2.8 ± 0.44
Ti-MAO 0.82 ± 0.16 2.37 ± 0.40 3.86 ± 0.64 0.60 ± 0.12 3.35 ± 0.49 –
Z Ti-MAO 0.58 ± 0.22 1.66 ± 0.58 2.75 ± 0.84 0.94 ± 0.11 4.25 ± 0.35 –
R. Gabo , L. C c
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6
ing ime [43]. The dis ibu ion o elemen s in he Ti-MAO coa ing is
shown in Fig. 4, and EDX analysis con i med he p esence o Ti, Si
and O elemen s (Table 3). Inc eased Si con en s we e de e mined
mainly in he ou e po ous laye , acco ding o Gabo e al.[36].
Acco ding o [44], he MAO p ocess in alkaline condi ions wi h
he p esence o silica es p o ides be e wea esis ance and co o-
sion esis ance. The p opo ion o silicon in he coa ing olume di -
e ed signi ican ly o MAO coa ings - he Z Ti-MAO silicon con en
(Fig. 5) de e mined by EDX was lowe in he coa ing olume
(Table 3). On he con a y, he Si con en s on he su aces o he
ou e MAO laye s abou 20 nm in hickness we e ela i ely close
due o he sensi i i y o he XPS me hod (Table 4). The educ ion
Fig. 4. C oss-sec ion SEM images o he Ti-MAO coa ing (a) and elemen al mappings o Ti, Si, O (b-d).
Fig. 5. C oss-sec ion SEM images o he Z Ti-MAO coa ing (a) and elemen al mappings o Z , Ti, Si, O (b-e).
Table 3
EDX esul s analysis o he Ti-MAO and Z Ti-MAO coa ings.
Sample Elemen (in a .%)
Ti Si Z O
Ti-MAO 9.4 ± 0.2 13.9 ± 0.2 – 76.7 ± 0.3
Z Ti-MAO 2.5 ± 0.1 3.2 ± 0.1 18.3 ± 0.6 76.0 ± 0.2
R. Gabo , L. C c
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7
o he silicon con en in he ou e laye is p obably associa ed wi h
he mig a ion o Z ions owa ds he su ace and he limi ed ans-
e o he silicon species om he elec oly e o he dep h o he
coa ing [45]. This ac is ela ed o he de elopmen o so-called
‘‘so ” spa king, which is e lec ed in he high densi y o blue-
whi e mic o-discha ges a e he o e all educ ion o discha ges
o e he en i e sample a ea and noise educ ion [46]. The esul ing
‘‘so ” spa king is p obably ela ed o he occu ence o small diam-
e e discha ge channels and he associa ed limi ed mass ans e
and educed silicon con en in he olume o he ou e po ous laye
[47].
Bo h he composi ion o he su ace and i s chemical s a e we e
moni o ed by he XPS me hod. The esul s o he semi-quan i a i e
analysis de e mined by he XPS me hod on indi idual samples (Ti-
MAO, Z Ti-MAO) a e shown in Table 4. The con en s o he indi id-
ual elemen s we e de e mined om he peak a ea by he me hod
o ela i e sensi i i y ac o s a e sub ac ing he Shi ley back-
g ound. The p esence o Ti, O, C, Si and Na was egis e ed on he
su ace o bo h samples. The p esence o Z om PVD deposi ion
(Z Ti) was also de e mined on he su ace o he Z Ti-MAO sample.
Elemen s Si, Na and C we e inco po a ed in o he su ace o he
MAO laye o he elec oly e.
The chemical s a e o indi idual elemen s was de e mined om
he peak shi s o indi idual elemen s. The axis o binding ene gies
–E
B
– was calib a ed unde he assump ion ha he main compo-
nen o he peak C 1s lies a E
B
= 282.5 eV and co esponds o he
Si-C bond [48]. A second componen was also de e mined a peak C
1s (Fig. 6), which has an in ensi y o app oxima ely 15% o he o al
signal C and lies a E
B
= 285.7 eV. Acco ding o Wang e al.[48], his
binding ene gy co esponds o he C-O g oups. On he su ace o
he Ti-MAO sample, wo componen s o he Si 2p peak a
E
B
= 99.7 and 101.0 eV we e de ec ed. Acco ding o Wang e al.
[49], his co esponds o he s uc u e o Si-C and C-O-Si. On he
Z Ti-MAO coa ing, h ee componen s o he Si 2p peak we e egis-
e ed, namely E
B
= 97.7, 98.7 and 100.00 eV. The e is p obably an
in e ac ion o Si wi h Z (Fig. 6), which is e iden om he shi
o E
B
o lowe alues by app oxima ely 1 eV. The peaks a
E
B
= 98.7 and 100.0 eV can bo h be iden i ied as o he Ti-MAO
sample, and co espond o Si-C and C-O-Si s uc u es. The spec-
um also has a peak a E
B
= 97.7 eV. This alue is compa able o
E
B
= 99.3 eV, epo ed in a s udy by Ding e al.[50] o he Z -Si
s uc u e.
The o e all s uc u e o Si can be shi ed o E
B
lowe by 1 eV.
This peak can also be a ibu ed o he Si-Si s uc u e. Shi s o
he Si 2p peaks o lowe binding ene gies can be caused by a cha ge
ans e om Z o Si and o he elemen s, because Z has lowe
elec onega i i y (Pauling elec onega i i y 1.33) han Si (1.90)
and o he elemen s. This causes a educ ion in he measu ed bind-
ing ene gy o he elemen o which he cha ge shi s mo es. The O
1s oxygen peak (Fig. 6) o he Ti-MAO sample su ace is wide, and
can be di ided in o wo componen s a ene gies o 529.3 and
529.9 eV. On he su ace o he Z Ti-MAO coa ing, ano he compo-
nen a E
B
= 527.7 eV was iden i ied. All hese peaks can be
explained by an oxidic bond, because he ange o oxygen binding
ene gies in oxides is ela i ely wide (528.1 – 531.1 eV) [51]. These
a e p obably he e o e oxides o Si, Ti and Z . The Ti 2p peak on he
Ti-MAO sample lies a E
B
= 456.9 eV. Due o he ene gy dis ance o
he Ti 2p
3/2
and O 1s peaks o 72.8 eV, we conclude ha Ti
2
O
3
is
p esen [52].
In bo h samples (Ti-MAO, Z Ti-MAO), he ex ension o he Ti 2p
and O 1s spec a is e iden . This sugges s he p esence o a mix u e
o lowe Ti oxides and, a he same ime, i co esponds o he mea-
su ed O 1s binding ene gies. In he spec um o zi conium Z 3d
5/2
(Fig. 6) on he su ace o he Z Ti-MAO sample, we ind wo com-
ponen s a E
B
= 177.6 and 179.2 eV. The peaks a e widesp ead
and he saddle be ween componen s 3d
5/2
and 3d
3/2
is almos
Table 4
XPS analysis esul s o MAO coa ings.
Sample Elemen (in a .%)
Ti O C Si Na Z
Ti-MAO 5.7 65.9 8.2 18.9 1.3 0.0
Z Ti-MAO 1.6 65.2 8.0 15.3 1.6 8.4
Fig. 6. XPS spec a o MAO coa ings.
R. Gabo , L. C c
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8
indis inguishable, indica ing high dispe sion o he Z p esen in
he laye . Acco ding o Ding e al.[50], who s udied he e ec o
Z Si
2
pa icles on phenol deg ada ion, he de e mined binding
ene gy E
B
= 179.2 eV co esponds o he Z /Si s uc u e. The com-
ponen wi h a lowe binding ene gy E
B
= 177.6 eV could no be
iden i ied acco ding o he spec um a las [51]. The XPS spec um
o Z 3d
3/2
and Z 3d
5/2
o he Z Ti-MAO sample is p obably in lu-
enced by he a omic dispe sion o zi conium and/o by he change
in he coo dina ion numbe o zi conia o o m a complex oxide
phase, which appea s o occu on he sample su ace [53]. The Z
3d
5/2
binding ene gy alues ha we e de e mined a e lowe han
o pu e Z O
2
(182.5 eV). The Na 1 s peak a 1070.2 and
1067.9 eV binding ene gies was also egis e ed on he su aces o
he sample (Ti-MAO, Z Ti-MAO).
The we abili y o he su aces was e alua ed using he de e -
mined con ac angles (CA) o he subs a e and o he MAO coa ings
(Supplemen a y Fig. S1). Compa ed o he subs a e, which had
CA = 89.07°± 3.28, he de e mined CA o he modi ied MAO coa -
ings showed hyd ophilic p ope ies co esponding o CA = 72.32°±
4.15 o he Ti-MAO coa ing and CA = 69.01°± 5.63 o he Z Ti-MAO
coa ing. The esul s con i m he well-known ac ha MAO coa -
ings, wi h hei po ous s uc u e and g ea e oughness, a e mo e
hyd ophilic han unmodi ied subs a es [54].
3.2. Wea beha io and coa ing adhesion
The cou se o he coe icien o ic ion was i s es ed on he
Ti-6Al-4V base ma e ial wi hou su ace ea men . Subsequen ly,
his was compa ed wi h he cou se o he coe icien o ic ion
o samples wi h a coa ing o pu e Ti-PVD and Z Ti-PVD wi hou
and wi h MAO ea men . Fig. 7 shows ha , a e unning-in, he
coe icien o ic ion o he MAO samples is e y s able, and ha
adhesi e ic ion p edomina es. In he base ma e ial and in pu e Ti
and Z Ti me als, howe e . he cou se o he coe icien o ic ion is
less s able, and a combina ion o adhesi e and ab asi e ic ion
p e ails. The a e age coe icien o ic ion o he es ed samples
is e y simila , and anges om 0.4 o 0.5, excep o he Z Ti-
MAO coa ing, o which he ic ion coe icien dec eased o 0.3.
The wea a e (Fig. 9b) was de e mined om he wea ack
p o ile (Fig. 8), and he wid h o he wea ack was used as an
addi ional pa ame e o wea compa ison (Fig. 9a). Fo he Ti-
PVD and Z Ti-PVD samples, he measu ed alues we e compa able
o he alues o he Ti-6Al-4V alloy. Bo h he Ti-PVD coa ings and
he Z Ti-PVD coa ings we e ab aded o he Ti-6Al-4V base ma e ial.
This is also con i med by he analysis o he chemical composi ion
o EDX a e ibological es s in PBS p esen ed in Table 6. The
chemical composi ion in he middle o he ace is compa able o
he chemical composi ion o he Ti-6Al-4V base ma e ial.
In con as , a signi ican dec ease in he wea a e was con-
i med o he MAO coa ings. Only he ou e laye (Fig. 8) o he
Ti-MAO coa ing was ab aded. This is con i med in Table 6, whe e
he e is a clea dec ease in Si in he middle o he ic ion ace.
The amoun o Si d opped om 21.0 w % o 3.8 w %.
Howe e , he Z Ti-MAO coa ing clea ly showed he bes wea
esis ance p ope ies. The ou e laye was minimally ab aded a
he poin o ic ion (Fig. 8). This is con i med by a compa ison
be ween he chemical composi ion in he middle o he wea ace
and on he ou side o he wea ace, which is unchanged (Table 5).
The Z Ti-MAO sample con i med low ic ion and e y good
wea esis ance in compa ison wi h he Ti-MAO sample (Fig. 9
and Table 5), and also in compa ison wi h he MAO sample (basic
Ti-6Al-4V ma e ial modi ied wi h MAO), which was es ed in ou
p e ious wo k [36].
Because only a small amoun o su ace wea was de ec ed on
he Z Ti-MAO sample, he chemical and bonding composi ion anal-
ysed by XPS can be used o explain his signi ican di e ence in
Fig. 7. A compa ison o he ic ion coe icien s o he Ti-6Al-4V, Ti-PVD and Z Ti-PVD samples (a), he Ti-MAO and Z Ti-MAO samples (b), and hei unning-in phases (c, d).
R. Gabo , L. C c
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