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Jou nal o Biomedical Ma e ials Resea ch Pa A (2013) n.101A
DOI: 10.1002/jbm.a.34489
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“This is he pee e iewed e sion o he ollowing a icle:
Lopez-San os, C.; Colaux JL, Laloy J; F ansole , M; Mullie , F; Michiels, C;
Dogné J-M, Lucas, S. 2013. Bioac i i y and hemocompa ibili y s udy o
amo phous hyd ogena ed ca bon coa ings p oduced by pulsed magne on
discha ge. J Biomed Ma e Res Pa A (2013):101A:1800–1812 which has been
published in inal o m a h ps:// doi.o g/ 10.1002/jbm.a.34489. This a icle may
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1
BIOACTIVITY AND HEMOCOMPATIBILITY STUDY OF AMORPHOUS
HYDROGENATED CARBON COATINGS PRODUCED BY PULSED
MAGNETRON DISCHARGE
C. Lopez-San os
1
, J.L. Colaux
1
, J. Laloy
2
, M. F ansole
3
, F. Mullie
2
,
C. Michiels
3
, J.-M.
Dogné
2
, S. Lucas
1
1) Namu Resea ch Ins i u e o Li e Sciences (NARILIS), Resea ch Cen e o he
Physics o Ma e and Radia ion (PMR), Uni e si y o Namu (FUNDP), Rue de
B uxelles, 61, B-5000 Namu , Belgium
2) Namu Resea ch Ins i u e o Li e Sciences (NARILIS), Namu Th ombosis and
Hemos asis Cen e (NTHC), Depa men o Pha macy, Uni e si y o Namu (FUNDP),
Namu , Belgium
3) Namu Resea ch Ins i u e o Li e Sciences (NARILIS), Resea ch Uni on Cell Biology
(URBC), Uni e si y o Namu (FUNDP) Rue de B uxelles, 61, B-5000 Namu , Belgium
Abs ac
Li e a u e con ains e y ew da a abou he po en ial biomedical applica ion o
amo phous hyd ogena ed ca bon (a-C:H) hin ilms deposi ed by eac i e pulsed
magne on discha ge e en so i is one o he mos scalable plasma deposi ion echnique.
In his pape , we show ha such a C
2
H
2
pulsed magne on plasma p oduces high quali y
coa ing wi h good hemocompa ibili y and bioac i e esponse: no e ec on hemolysis and
hemos asis we e obse ed, and p oli e a ion o a ious cell ypes such as endo helial,
ib oblas and os eoblas -like cells was no a ec ed when he deposi ion condi ions we e
Page 1 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A
2
a ied. Cell g ow h on a-C:H coa ings is p oposed o ake place by a wo-s ep p ocess:
he ini ial cell con ac is a ec ed by he smoo h opog aphy o he a-C:H coa ings,
whe eas he polyme ic-like s uc u e, oge he wi h a mode a e hyd ophilici y and a high
hyd ogen con en , di ec s he pos e io cell sp eading while p ese ing he
hemocompa ible beha io .
Keywo ds: cy ocompa ibili y, hemocompa ibili y, a-C:H coa ing, polyme ic-like
s uc u e, C
2
H
2
magne on discha ge
In oduc ion
Amo phous hyd ogena ed ca bon (a-C:H) hin ilms a e in e es ing ma e ials due o he
singula s uc u e composed o ca bon bonded in sp
2
and sp
3
elec onic con igu a ions
and hyd ogen
1
. Fi s ly, hei special p ope ies, such as a good ha dness, high wea
esis ance, chemical ine ness and low ic ion coe icien make hem e y use ul as
p o ec i e coa ings. Mo eo e , good bio- ole ance oge he wi h he absence o
cy o oxici y make hem sui able o biomedical applica ions
2
. As a esul , medical
de ices such as medical guidewi es, su gical needles, in aocula lenses, co ona y s en s
and hea al es a e co e ed wi h a-C:H coa ings
3
. O he simila hin ilms like diamond-
like coa ings (DLC) a e commonly used in o hopaedic de ices as hip, ascula and
den al p os heses
4-9
, showing he e sa ili y o hese ca bon-based ma e ials.
The e a e se e al me hods o he deposi ion o a-C:H coa ings: plasma-enhanced
chemical apou deposi ion
10
, ion beam deposi ion
11
o pulsed lase abla ion
12
. Recen ly,
he pulsed eac i e magne on p ocess has a ac ed a high in e es in many indus ial
Page 2 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A
3
applica ions
13,14
. The e sa ili y o his deposi ion me hod allows o ine une he
chemical composi ion o he a-C:H coa ings ha is usually equi ed o ensu e a good cell
g ow h. Fo example, while ib oblas cells do no equi e special uning o p oli e a e on
a-C:H ma e ial deposi ed by lase o plasma echniques
15,16
, os eoblas cell g ow h is
imp o ed by he inco po a ion o elemen s like i anium
3,17
, bo on
18
o silicon
19
.
Mo eo e , good bioac i i y esponse o endo helial cells has been obse ed a e doping
wi h ni ogen o silicon o such ma e ial g own by plasma sys ems
3,20-23
.
Biocompa ibili y is no only dependen on he chemical composi ion since o he physico-
chemical p ope ies such as he we abili y, pola i y o oughness a e also o
impo ance
3,6
. Fo example, an hyd ophilic su ace seems o enhance he adso p ion o
se e al cell-adhesion media ing p o eins, hence cell adhesion i sel
3
and he su ace
pola i y can imp o e cell p oli e a ion
24-26
. Su ace opog aphy is one o he c i ical
ac o s because i has an in luence on cell-ma e ial in e ac ions. Thus, in- i o s udies
ha e shown ha os eoblas -like cells a ach p e e en ially o su aces wi h a oughe
mic o opog aphy
27
. The e o e, he bioac i e esponse o he ma e ial may be dependen
on he cell line as he same su ace p ope ies can be an ad an age o a p oblem o he
cell g ow h and p oli e a ion. This ac could limi he po en ial biomedical applica ions
o he a-C:H coa ing.
I one hinks o use a-C:H coa ings o po en ial applica ions in ol ing con ac wi h
blood, i mus ul il a leas h ee c i e ia o be hemocompa ible: no des uc ion o he ed
blood cells (RBCs) (hemolysis), no o limi ed impac on pla ele unc ion (adhesion,
ac i a ion and agg ega ion) and no induc ion on coagula ion (seconda y hemos asis). I is
usually accep ed ha he main pa ame e s a ec ing he hemocompa ibili y o a coa ing is
Page 3 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A
4
he we abili y o he su ace, modula ed by he su ace chemical composi ion and
oughness. Indeed, hyd ophilic coa ings ( e y low wa e con ac angle) a ou he
adso p ion o albumin agains ib inogen, which is sui able o he inhibi ion o h ombus
o ma ion
28-30
. Pa icula ly, hyd ophilic polyme ic-like ma e ials ha e also shown non-
h ombogenic p ope ies
31
. Bu li e a u e shows ha samples pa ially hyd ophilic, wi h
wa e con ac angles be ween 56-90°, p esen ed he bes hemocompa ible beha io
32,33
.
O he con adic o y examples a e also ound: non- h ombogenic p ope ies ha e been
obse ed o hyd ophobic diamond-like ca bon coa ings doped wi h luo ine o silicon
31
.
The e o e, he hemocompa ibili y o a-C:H coa ings is no only dependen on he su ace
we abili y. Especially, pla ele ac i a ion can be also a ec ed by he mo phology
29
and,
mo e ecen ly, i has been p oposed ha he le el o ca bon sp
3
/sp
2
hyb idiza ion o
doped a-C:H laye s has an in luence on he hemocompa ibili y h ough he pla ele
adhesion
34
.
This wo k aims a he e alua ion o he eac i e magne on spu e ing sys em o deposi
bioac i e a-C:H hin ilms. Li e a u e con ains only a ew biocompa ible and
hemocompa ible da a on he p ope ies o a-C:H laye g own by his echnique, despi e
he ac ha i is p obably one o he cheapes in ol ing plasma and wi h he bes
indus ial scalabili y. The e o e, we p esen he e a de ailed analysis o he in- i o
hemocompa ibili y (e ec s on hemolysis, pla ele unc ion and coagula ion) and bioac i e
esponses o endo helial, ib oblas and os eoblas -like cells on he a-C:H coa ings.
Finally, he cha ac e iza ion o he physico-chemical p ope ies o hese a-C:H coa ings is
s udied o con ibu e o he unde s anding o he ma e ial beha io and he de ini ion o
he sui able p ope ies o a po en ial biomedical applica ion.
Page 4 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A

5
Ma e ial and me hods
Coa ing deposi ion and cha ac e iza ion
The eac o consis s o a acuum chambe (<10
-4
Pa o esidual p essu e) equipped wi h
wo con ocal pulsed magne on spu e ing sou ces loaded wi h 2” g aphi e a ge s (99.9%
pu i y). A eac i e mix u e o a gon, ace ylene and hyd ogen gasses a a a io o 20:4:1
sccm was used o gene a e he eac i e plasma. Di e en alues o powe densi y om 7
o 21 W cm
-2
we e applied simul aneously on bo h a ge s (one powe supply pe
magne on) in cu en mode con igu a ion. The p essu e was main ained a 0.4 Pa du ing
50 min o deposi ion, whe eas he pulse-on and pulse-o imes we e bo h se o 50 µs.
Well cleaned glass co e slips o 13mm diame e , h ough ul asonic ba hs wi h bi-
dis illed wa e and washing wi h e hanol, we e used as subs a es.
The a-C:H composi ion dep h p o iles we e de e mined by ion beam analysis combining
Elas ic Recoil De ec ion Analysis (ERDA) and Ru he o d Backsca e ing Spec oscopy
(RBS), using an impinging beam o
4
He a 2.2 MeV and a pos e io spec a i ing wi h
SIMTa ge
35
and SIMNRA
36
codes. X- ay pho oelec on spec oscopy (XPS) s udy was
pe o med in an SSX-100 spec ome e (Su ace Science Ins umen s) wi h an Al K
α
adia ion a 1486.6 eV as exci a ion sou ce. Raman spec oscopy was ca ied ou by a
LabRAM HR High Resolu ion 800 UV Con ocal Raman mic oscope wi h a g een lase
(He-Ne 532.14 nm), in backsca e ing con igu a ion.
Thickness o he a-C:H laye was measu ed wi h a s ylus p o ilome e om Veeco
Ins umen s (Su ace P o ile Measu ing Sys em Dek ak). Su ace opog aphy was
analyzed by a Mul imode 8 AFM (Veeco diNanoScope V), wo king in apping mode,
wi h Si ips o 350-380 kHz esonan equency and 8-20 N/m sp ing cons an .
Page 5 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A
6
T ibological analysis was done in a Hysi on Inc. T iboinden e sys em wi h he load and
displacemen esolu ions o 0.1 µN and 0.1 nm, espec i ely. Ha dness and educed
Young’s modulus we e es ima ed using a Be ko ich inden e wi h a ace angle o 65.3°.
We abili y s udy was pe o med h ough s a ic wa e con ac angle measu emen s wi h
OCA20 equipmen and SCA20 so wa e (Da aphysics), acco ding o he Young’s
me hod
37
. Bidis illed wa e sessile d ops o 1 µl we e employed. Young’s me hod ela es
he angle o a liquid d ople in con ac o a solid su ace wi h he in e acial ensions a
equilib ium. The epo ed alues a e an a e age o en measu emen s aken o each
examined su ace. To es ima e he su ace ension o he a-C:H coa ing, diiodome hane
con ac angles we e measu ed in a simila way, acco ding o he Owens-Wend -Kaelble
app oxima ion
38
. This model p esen s a su ace ension composed o wo con ibu ions:
one due o pola in e ac ions and one due o non-pola o dispe si e in e ac ions be ween
he liquid and he solid phases. The e o e, he measu emen o con ac angles wi h a leas
wo liquids is equi ed, whe e diiodome hane is chosen o emphasize he non-pola
in e ac ions unlike wa e
39,40
.
Biocompa ibili y es
- Cell cul u e and iabili y assay
Samples we e s e ilized in an au ocla e (20 min a 121 °C and 1,2 x10
5
Pa). Pos e io
XPS and we ing measu emen s ha e con i med ha he e we e no signi ican changes
a e s e iliza ion: only a sligh inc ease o 3 % o he oxygen su ace concen a ion o a-
C:H and a sligh inc ease in he wa e con ac angle o abou 3° we e ound. These
a ia ions can be conside ed wi hin he e o ma gins o he echniques. EAhy926 human
Page 6 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A
7
endo helial cell line was seeded on he samples a a densi y o 10,000 cells/well in 0.5 ml
o he cul u e medium Dulbecco’s Modi ied Eagle Medium (DMEM, Gibco)
supplemen ed wi h 10% ( : ) e al bo in se um (FBS, Gibco, US) in 24-well pla es.
Endo helial cells we e main ained in cul u e a 37 °C unde 5 % CO
2
and incuba ed om
1 o 3 days. A each day, he numbe o iable cells was de e mined wi h a MTT assay
using Thiazolyl Blue B omide MTT (2.5 mg/ml o [3-(4.5-dime hyl hiazol-2-yl)-2.5-
diphenyl e azolium b omide], Sigma-Ald ich , Ge many, in Phospha e bu e ed saline
(PBS), 6.7 mM phospha e, pH 7.4) which allows measu ing he cell iabili y on a-C:H
coa ings. 500 µl o MTT solu ion we e added o he medium o 2 h a 37 °C in a 5% CO
2
a mosphe e. Fo mazan c ys als a e lysed in a "lysis bu e " composed o SDS 30% in
H
2
O
d
+ N,N-dime hyl- o mamide 2:1 ( : ) a pH: 4.7. The op ical abso bance was
measu ed wi h a pla e spec opho ome e (xMa k
TM
, Bio-Rad, US) a 570 nm. MTT
p o ocol is also applied o wo o he cell ypes. The human skin ib oblas AG04431
(Co iell Ins i u e o Medical Resea ch, USA) and he human MG-63 os eoblas -like cells
(ATCC, USA), we e espec i ely g own in Basal Medium Eagle (BME, Gibco, US)
supplemen ed wi h 10 % FBS and 2 mM L-glu amine and in DMEM supplemen ed 10 %
FBS. S a is ical analysis was ca ied ou using one-way analysis o a iance (ANOVA),
p esen ing he mean alue om 3 epea s and he e o ba s e e ing o he s anda d e o
o he mean. In wha ollows, glass subs a e was used as posi i e con ol because o he
good cell adhesion and sp eading on i . Meanwhile a coppe (Cu) hin ilm deposi ed in
he same sys em han a-C:H coa ings was used as nega i e con ol because o i s
cy o oxici y.
Page 7 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A
8
- Cell mo phology s aining
Cell mo phology was obse ed a e cell p o ein s aining wi h Coomassie Blue. Cells
we e washed wice wi h PBS, and fixed wi h 3.7% pa a o maldehyde (PFA) in dis illed
wa e o 10 min a oom empe a u e (RT). Subsequen ly, hey we e pe meabilized wi h
70% e hanol in dis illed wa e o 10 min a RT and d ied a 37 °C. The cells we e finally
s ained wi h Coomassie Blue o 30 s, washed wi h dis illed wa e and d ied a 37 °C,
be o e being obse ed wi h an in e ed mic oscope (Labo e FS, Lei z). In o de no o
epea simila ob ained in o ma ion, only EAhy926 endo helial cells mo phology s aining
is p esen ed in he nex Resul s sec ion.
- Cy oskele on analysis
Cy oskele on mo phology was s udied in cells by immuno luo escence s aining. As
example o he simila esul s, we p esen ed only he images co esponding o he
EAhy926 endo helial cells. Cells we e ixed wi h 4 % PFA o 10 min a 37 °C.
Subsequen ly, he samples we e washed wi h PBS and pe meabilized wi h 1 % T i on-X-
100 (Sigma-Ald ich , Ge many). Ac in was s ained wi h Alexa Fluo 546 phalloïdin
(Molecula P obes) and nuclei wi h To-P o-3 (Molecula P obes) dilu ed (1:80) in PBS-
RNase (2 mg/ml). The samples we e again washed h ee imes be o e being embedded in
Mowiol (Ald ich) and pho og aphed wi h a con ocal mic oscope TCS (Leica SP1) using
a 409 objec i e and a cons an pho omul iplie .
- In lamma o y cy okine sec e ion
Page 8 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A
15
Cell g ow h analysis
Cell adhesion was e alua ed a he 24h a e seeding on he a-C:H coa ings. Coomassie
blue s aining, p esen ed on he endo helial cells as example, co obo a ed he good
a achmen and cell sp eading on he a-C:H su ace (Fig. 3). Fu he mo e, ac in
cy oskele on mo phology was obse ed 24 h a e seeding o he endo helial cells on he
di e en coa ings (Fig. 4). Bo h on he posi i e con ol and on he di e en a-C:H
coa ings, cells p esen ed a well sp ead wi h a well-s uc u ed cy oskele on wi h he
de elopmen o ac in ibe s, es ablishing an o ganized ibe s meshwo k dis ibu ed
h oughou he cell body and p e e en ially o ien ed along he main nucleus axis.
Cell p oli e a ion was ollowed h ough MTT assay 2 and 3 days a e seeding. The small
alues a e due o he ac ha a low cell densi y was seeded due o he small size o he
samples, ying o a oid he cell con luence a 3 days o cul u e. The a-C:H hin ilms did
no induce any de imen al o oxic e ec o EAhy926 endo helial, AG04431 skin
ib oblas s, o MG63 os eoblas -like cells a 2 days o cul u e, as can be obse ed in Fig.
5. The esul s demons a e ha he e we e as much iable cells on a-C:H coa ings as on
he posi i e con ol, while cells seeded on he nega i e con ol we e a ec ed by oxici y.
The a-C:H coa ings had no s a is ical e ec s compa ed o he posi i e con ol. Thus, he
op ical densi y alues om he a-C:H coa ings espec o hose co esponding o he
posi i e con ol, aking accoun he s a is ical e o ba s, a e e y simila . In he wo s
cases, hey ep esen mo e han he 85% o he posi i e con ol esul , wha is conside ed
a good bioac i e esponse. Also, well-sp eading o he cells on he a-C:H su ace was
obse ed, as ep esen ed he esul s om EAhy926 endo helial a e 3 days o seeding,
simila o hose om he o he cul u ed cells. Cell mo phology on he a-C:H su ace was
Page 15 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A

16
simila o he one obse ed o he posi i e con ol and opposi e o he nega i e con ol,
as shown in Figs. 6 - 8. Indeed, he e was no su i al o any o he h ee ypes o cells
when seeded on he nega i e con ol su ace.
Addi ionally, IL-8 p oduc ion is known o be caused by cells unde going on
in lamma o y esponse
48
. As obse ed in Fig. 9, cells seeded on he di e en coa ings did
no p oduce mo e IL-8 han he cells seeded on glass (posi i e con ol). This indica es
ha no in lamma ion esponse was aking place on he a-C:H coa ings.
Hemocompa ibili y es
The e ec o a-C:H coa ing deposi ed a he in e media e powe densi y, on he lysis o
human RBC, is p esen ed in Table 3. The a-C:H coa ing as well as he o he nega i e
con ols showed insigni ican hemoly ic e ec (< 5 % in compa ison o he posi i e
con ol)
49
.
P ima y hemos asis analysis h ough he s udy o pla ele unc ion (adhesion, ac i a ion
and agg ega ion) in Fig. 10A e eals he adhesion o only ew pla ele s on he a-C:H hin
ilm. Adhe en pla ele s emained no ac i a ed on he a-C:H su ace and kep hei
con o ma ion (absence o pseudopodia). No pla ele agg ega ion was obse ed.
Fig. 10B shows he esul s o he cTGT on he a-C:H coa ing acco ding o
41
, compa ed o
nega i e and posi i e con ols. A e 1 h o incuba ion wi hou 4 µM o PL induce , i s
aces o ac i e h ombin appea ed in NPP a e a lag ime o 17 min. The C
max
was
a ound 70 nM and was used as e e ence o he ollowing esul s. Con e sely, glass
sample used as posi i e con ol e ealed a ema kable inc ease in he ca aly ic ac i i y o
h ombin (inc ease o C
max
o 148 nM and educ ion o lag ime o 13 min), indica ing
Page 16 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A
17
ac i a ion o coagula ion. The h ombin gene a ion p o ile o he a-C:H hin ilm
appea ed e y simila o he one o he nega i e con ol (i.e. silicon) and o he NPP
plasma cu es.
Discussion
The special physico - chemical p ope ies o a-C:H coa ings ha e p e iously been
associa ed o a good p o ec i e unc ion, aising he in e es in po en ial biomedical
applica ions, as p esen ed in he in oduc ion sec ion. Recen ly, he eac i e magne on
p ocess has been ecognized as a well es ablished p oduc ion sys em o a-C:H coa ings in
many indus ial ields. The e o e, a-C:H coa ings deposi ed by eac i e magne on
spu e ing o a biomedical unc ion mus be biocompa ible as well as hemocompa ible o
ensu e good li ing cells - ma e ial in e ac ions. Mo eo e , he possibili y o ob ain
di e en su ace p ope ies o a-C:H coa ings makes in e es ing he analysis o he
in luence o pa ame e s such as he chemical composi ion, s uc u e and opog aphy in
hei bioac i e esponse. We showed ha he p esen a-C:H coa ings con ain mo e han
30 a . % o hyd ogen and exhibi a I
D
/I
G
a io be ween 0.77 - 0.92 and a e y low
oughness. The a-C:H coa ings p esen a mode a e hyd ophilic cha ac e (wa e con ac
angles a ound 40 - 74°) and he su ace- ension s ays below 62 mN/m. The biop ope ies
o such su aces a e a good cell p oli e a ion ( ib oblas s, endo helial cells and os eoblas -
like cells) and hemocompa ibili y (no hemolysis, p e en ion o pla ele
adhesion/ac i a ion/agg ega ion, and p e en ion o h ombin gene a ion). Ou discussion
o he ela ionship be ween he bioac i e esponse and he physico-chemical p ope ies
Page 17 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A
18
will be p esen ed in ligh o : i) we abili y and su ace ension, ii) su ace opog aphy and
iii) su ace s uc u e.
I) We abili y and su ace ension
The we ing beha io o he ma e ial has al eady been ela ed o he cell p oli e a ion. Fo
example, Van Wachem e al.
50
ha e shown ha a mode a e hyd ophilici y o polyme ic
su aces imp o ed he adso p ion o se um p o eins, a ec ing posi i ely he cell
a achmen and sp eading. C espin e al.
48
ha e con i med ha endo helial cell densi y
was highe on a-C:H coa ings wi h wa e con ac angle alues be ween 40 - 75°. This
endency is in ag eemen wi h he we abili y obse ed o he p esen a-C:H coa ings.
Pola oxygen species in he a-C:H coa ing ha e been sugges ed o be esponsible o he
induced hyd ophilici y. The p esence o oxygen pola g oups on he a-C:H su ace would
acili a e cell adhesion h ough ionic in e ac ions wi h unc ional g oups such as NH
2
and
COOH om cell p o eins, ha a e posi i ely o nega i ely cha ged a physiological pH,
espec i ely
43,44
. These oxygen pola g oups a ec he pola componen alue o su ace
ension o he a-C:H coa ings, a ound 4 - 23 mN/m, and consequen ly he pola i y. Hallab
e al.
51
ha e also p oposed ha pola i y alues below 0.5 a e bene icial o he cell
adhesion, which is he case o he a-C:H coa ings as desc ibed in he Table 2. Thus,
esul s om Tables 1 and 2 show ha he a-C:H coa ings exhibi a change in he wa e
con ac angle and in he pola componen o he su ace ension wi hou a change in he
in-dep h oxygen con en bu only in he su ace composi ion, he inc ease o he powe
densi y acili a ing he gene a ion o oxygen-con aining g oups a he su ace o he
coa ing.
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19
Ano he con ibu ion o ake in o accoun is he educ ion o he wa e con ac angle due
o he p esence o anspolyace ylene chains in he mic os uc u e
52
, since hey a o he
inco po a ion o pola oxygen unc ional g oups o he su ace. Mo eo e , he in luence
o he oughness on he we abili y can be obse ed om Fig. 2 and Table 2, whe e a
lowe wa e con ac angle is associa ed wi h a oughe su ace. Finally, Fig. 11A ies o
co ela e he cell iabili y and he hyd ophilici y and, indi ec ly he su ace oxidiza ion o
he a-C:H coa ings. We abili y as well as su ace ension and su ace oxygen con en
p o ided he same ela ionships o he iabili y o he h ee cell lines. Howe e , s a is ical
e o o he cell iabili y esul s does no allow o conside clea endencies be ween he
g ow h o each cell line on he samples and hei hyd ophilici y. Only o he case o
MG63 cells could be hough abou an imp o emen o he cell iabili y on a su ace wi h
a lowe wa e con ac angle.
I has also been shown ha we abili y and su ace ension ha e also a s ong in luence on
he hemocompa ibili y o a-C:H hin ilms. A low hemolysis a e has been ela ed o a
hyd ophilic su ace ha would imp o e he compa ibili y o biofluids
29,53
. When blood
en e s in con ac wi h he a-C:H coa ing, plasma p o eins a e adso bed on he su ace and
pla ele s adhe ed o he plasma-p o ein coa ed ma e ial. Fib inogen plays a majo ole in
pla ele agg ega ion and ib in o ma ion in coagula ion, and subsequen ly in he
o ma ion o h ombus. Roy e al.
29
ha e men ioned ha when he elec os a ic in e ac ion
be ween a nega i ely cha ged hyd ophilic su ace and he posi i ely cha ged domains o
ib inogen is weak, he e is a educ ion o he ib inogen adso p ion and he pla ele
adhesion. Also, i has been ound ha pla ele s we e less a ached on hyd oxyl-con aining
polyme su aces
28
. Howe e , o he wo ks ha e p esen ed a low hemolysis a e in
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20
hyd ophobic polyme s
54
. In simila ma e ials like DLC coa ings, wa e con ac angles
a ound 60 - 70° esul ed in he educ ion o he plasma p o eins adso p ion. Thus, an
in e acial ension be ween he coa ing and albumin lowe han be ween he coa ing and
blood o ib inogen, educes he deg ada ion o p o ein s uc u e and he ac i a ion o
blood coagula ion mechanism
30
.
II) Su ace opog aphy
In gene al, i is known ha oughness p omo es a good e ec on he cell ancho age on
polyme ic ma e ials
51,55,56
. Ne e heless, ib oblas s adhe ed be e on smoo he
su aces
2,57
. Acco ding o he p esen esul s, i is no possible o hink abou a di ec
co ela ion be ween he inc ease in he cell iabili y and he oughness o he a-C:H
coa ings as Fig. 11B shown. A he low le els o smoo hening shown by he a-C:H
su aces, cell iabili y is p ac ically independen o any cell line. E en i a be e MG63
cell iabili y could be suspec ed in oughe samples, he s a is ical e o canno ensu e i .
On he con a y, su ace opology, i.e. he shape o opog aphic ea u es, has an in luence
on he cell adhesion
48,58,59
. Typical dimensions o biocompa ible nanos uc u es ha e
been ound o ha e a heigh o abou 10 nm and a la e al spacing below 75 nm
60
. Fo
example, su aces wi h opog aphic ea u es o low dimension (less han 100 nm) a e
sui able o he adhesion o os eoblas cells, since hey can mimic he s uc u e o na u al
issues o p o eins and ex acellula pa s (wi h dimensions a ound ens o nanome e s)
3
.
The p esen esul s ha e showed he gene a ion o ea u es on he a-C:H su ace wi h
dimensions in a simila ange, jus i ying he gene alized good bioac i e esponse.
Smoo h opog aphies ha e also imp o ed he hemocompa ible beha io , h ough he
plasma p o eins abso p ion. The size o human se um albumin molecule is a ound 3x8
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21
nm
2
, whe eas o he ib inogen molecule is o 7x46 nm
2
. Fo his eason, su ace ea u e
dimensions mo e simila o he albumin size we e ound o a o i s abso p ion a he
han he one o ib inogen
61
.
III) Su ace s uc u e
This wo k links he cell sp eading on a-C:H coa ings deposi ed by eac i e pulsed
magne on spu e ing o he s uc u e o he coa ing (I
D
/I
G
a io) a he same le el o
impo ance han hose o he we abili y, he chemical composi ion o he opog aphy. A
p e ious wo k es ablished such a ela ionship o N-doped DLC and Si-doped DLC
coa ings
21
. In ha case, a high hyd ogen concen a ion oge he wi h a low I
D
/I
G
alue
(0.2 - 0.5) had a good in luence on endo helial cell adhesion. A he p esen case, he
I
D
/I
G
a io should be in he ange o 0.77 - 0.92 o ge a good cell p oli e a ion on a-C:H
coa ings wi hou any addi ional chemical elemen , as Fig. 11C indica es. I seems ha a
highe I
D
/I
G
a io, i. e. a mino ca bon sp
3
bonds concen a ion o a mo e o de ed a-C:H
s uc u e, a o he p oli e a ion o MG63 cells, unlike Eahy926 cells. Howe e , aking
accoun he ma gin o s a is ical a ia ion, i is no possible o ex ac a clea conclusion
abou endencies be ween he cell iabili y and he a-C:H s uc u e.
Recen ly, endo helial cell adhesion by a wo-s ep p ocess has been p oposed
62
: in he
ini ial cell con ac , he opog aphy is he main ac o o in luence, whe eas he su ace
chemis y go e ns he ollowing cell sp eading. An ex apola ion can be made o he
ib oblas and os eoblas -like cell g ow h on he deposi ed a-C:H coa ings. This wo s eps
p ocess is an in e es ing concep , bu has o be sligh ly modi ied in he iew o ou
indings: good cy ocompa ibili y esul s ha e been ob ained wi h di e en le els o
hyd ophilici y, su ace oughness and chemical composi ion. The e o e, we p opose ha
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22
in he wo s ep p ocess, cell sp eading is go e ned by he polyme ic-like s uc u e o ou
coa ings, desc ibed by he le el o sp
3
/sp
2
ca bon hyb idiza ion. Ma e ial s uc u e o he
a-C:H coa ings should ha e a s onge in luence in he cell p oli e a ion in o de o le el
he e ec s o he di e en su ace chemis y and oughness. O cou se, he mode a e
hyd ophilici y, h ough he oxygen inco po a ion, and he high hyd ogen con en a e
pa ame e s ha p edispose he su ace o a good bioac i e esponse. This p oposi ion is
also sui able o hemocompa ibili y. Indeed, he sp
3
concen a ion o he p esen a-C:H
coa ings is simila o hose p esen ed by Logo he idis e al.
61
who obse ed a high
abso p ion o albumin e sus ib inogen. Hemocompa ibili y o his ype o ilms became
wo se when sp
3
/sp
2
a io inc eased abo e 1.1 o he I
D
/I
G
a io was highe han 1.6
30,63
.
This is in line wi h ou sugges ion ha a sp
3
/sp
2
a io a ound 0.8 - 0.9 is sui able o cell
sp eading and o p e en h ombus gene a ion on he a-C:H su ace.
Conclusion
A good bioac i e esponse o a-C:H hin ilms deposi ed by eac i e pulsed magne on
spu e ing wi h endo helial, ib oblas and os eoblas -like cells ha e been ob ained. A he
same ime, he a-C:H coa ings we e hemocompa ible, wi hou any hemoly ic and
hemos as ic e ec s. Su ace chemical composi ion, oughness as well as we abili y
changes a e impo an pa ame e s, bu canno solely explain he obse a ions. Fo his
eason, we p oposed ha cell p oli e a ion occu s by a wo-s ep p ocess: in he ini ial cell
con ac , he smoo h opog aphy is he main ac o o in luence, whe eas he polyme ic-
like cha ac e , oge he wi h he su ace chemis y (mode a e hyd ophilici y h ough
Page 22 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A
23
oxygen inco po a ion and high hyd ogen con en ), go e ns he ollowing cell sp eading
and p ese es he hemocompa ibili y.
Acknowledgemen s
This wo k was pa ially suppo ed by he T ibo u u p ojec (Belgian Walloon Region
Funding), he REGPOT-CT-2011-285895-AL-NANOFUNC Eu opean p ojec and by he
CERUNA unding o he Uni e si y o Namu . We hank P . D. Boni azi and P . B.-L.
Su om he Dep . o Chemis y, Uni e si y o Namu (FUNDP), o he access o he
AFM and con ac angle equipmen s espec i ely, P . M.P. Delplancke-Ogle ee om he
Dep . o Indus ial Chemis y, F ee Uni e si y o B ussels (ULB), o he access o he
nanoinden e and D . J.C. Gonzalez om he Ins i u e o Ma e ials Science o Se ille
(ICMS) o he Raman measu emen s.
Page 23 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A
24
Re e ences
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Os eoblas s on Su ace-Modi ied Ma e ials De eloped o Bone Implan s.
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31
Lis o igu e cap ions
Fig. 1.- A) XPS spec a o he a-C:H coa ing deposi ed by eac i e pulsed magne on
spu e ing (12W cm
-2
); B) Raman spec a o he a-C:H coa ing deposi ed by eac i e
pulsed magne on spu e ing (12W cm
-2
).
Fig. 2- AFM opog aphy images (1µmx1µm) o he con ol samples used in he di e en
cy ocompa ibili y and hemocompa ibili y es s: A) glass co e slip, B) coppe hin ilm,
C) silicon hin ilm, and o he a-C:H coa ings a di e en applied powe densi y alues
and hickness: D) 7 W cm
-2
/ 200nm, E) 12 W cm
-2
/ 260nm, F) 21 W cm
-2
/ 454nm.
Heigh scale is indica ed in he g ey colo scale and he oughness is ep esen ed as he
mean squa e oughness coe icien R
ms
.
Fig. 3.- Mo phology s udy o EAhy926 endo helial cells adhesion on he a-C:H coa ings
analyzed by Coomassie Blue s aining and op ical mic oscopy 24 h a e cell seeding.
Resul s a e compa ed o hose o he posi i e (glass) and nega i e (Cu) con ols.
Fig. 4.- Ac in cy oskele on analysis o he EAhy 926 endo helial cells g ow h on he a-
C:H coa ings. Nuclei and ac in s ess ibe o ma ion we e obse ed in blue and ed,
espec i ely. Resul s a e compa ed o hose o he posi i e (glass) and nega i e (Cu)
con ols.
Fig. 5.- A) MTT iabili y es a 2 and 3 days a e EAhy926 endo helial cell seeding on
he deposi ed a-C:H coa ings; B) MTT iabili y es a 2 days a e ib oblas (FS) and
MG63 os eoblas -like cell seeding on he deposi ed a-C:H coa ings. Resul s a e exp essed
as mean ± 1 SD (n = 3). *; **; ***: p<0.05; p<0.01; p<0.001 e sus glass.
Fig. 6.- Cell mo phology o EAhy926 endo helial cells seeded on he a-C:H hin
deposi ed wi h di e en applied powe densi ies, a e 3 days o cell cul u e. Images we e
Page 31 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A

32
aken in phase con as mic oscopy. Resul s a e compa ed o hose o he posi i e (glass)
and nega i e (Cu) con ols.
Fig. 7.- Cell mo phology o ib oblas s seeded on he a-C:H hin deposi ed wi h di e en
applied powe densi ies, a e 3 days o cell cul u e. Images we e aken in phase con as
mic oscopy. Resul s a e compa ed o hose o he posi i e (glass) and nega i e (Cu)
con ols.
Fig. 8.- Cell mo phology o MG63 os eoblas -like cells seeded on he a-C:H hin
deposi ed wi h di e en applied powe densi ies, a e 3 days o cell cul u e. Images we e
aken in phase con as mic oscopy. Resul s a e compa ed o hose o he posi i e (glass)
and nega i e (Cu) con ols.
Fig. 9.- E alua ion o he p o-in lamma o y IL-8 cy okine sec e ion by EAhy926
endo helial cells cul u ed o 48 h on he a-C:H coa ings, compa ed o he posi i e (glass)
and nega i e (Cu) con ols. Resul s a e exp essed in pg o IL-8/µg o p o eins, as mean ±
1 SD (n = 3). *; **; ***: p<0.05; p<0.01; p<0.001 e sus glass.
Fig 10.- A) SEM images o he pla ele s on he a-C:H coa ing (12W cm
-2
). Scale ba es =
10 and 1 µm. On he le , homogenous epa i ion o pla ele s on he su ace; on he igh ,
iew o adhe en pla ele s; B) Calib a ed h ombin gene a ion es on he a-C:H coa ing,
compa ed o a posi i e con ol (glass), a nega i e con ol (Si) and he NPP plasma, using
he con ac pa hway h ombin induce . Cu es ep esen he mean o h ee indi idual
measu emen s.
Fig 11.- Cell iabili y esul s a 2 days o cell cul u e o he a-C:H coa ings in unc ion o :
A) hyd ophilici y (WCA), B) oughness (R
MS
) and C) s uc u e (I
D
/I
G
a io).
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