Bioactivity and hemocompatibility study of amorphous hydrogenated carbon coatings produced by pulsed magnetron discharge

Depósi o de in es igación de la Uni e sidad de Se illa h ps://idus.us.es/ Es a es la e sión acep ada del a ículo publicado en: This is a accep ed manusc ip o a pape published in: Jou nal o Biomedical Ma e ials Resea ch Pa A (2013) n.101A DOI: 10.1002/jbm.a.34489 Copy igh : 1999-2024 John Wiley & Sons El acceso a la e sión publicada del a ículo puede eque i la susc ipción de la e is a. Access o he published e sion may equi e subsc ip ion. “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 be used o non-comme cial pu poses in acco dance wi h Wiley Te ms and Condi ions o Use o Sel -A chi ed Ve sions. This a icle may no be enhanced, en iched o o he wise ans o med in o a de i a i e wo k, wi hou exp ess pe mission om Wiley o by s a u o y igh s unde applicable legisla ion. Copy igh no ices mus no be emo ed, obscu ed o modi ied. The a icle mus be linked o Wiley’s e sion o eco d on Wiley Online Lib a y and any embedding, aming o o he wise making a ailable he a icle o pages he eo by hi d pa ies om pla o ms, se ices and websi es o he han Wiley Online Lib a y mus be p ohibi ed." 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. Page 18 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A 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 Page 19 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A 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 Page 20 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A 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 Page 21 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A 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 1. Robe son J. Diamond-like amo phous ca bon. Ma e ials Science & Enginee ing R-Repo s 2002;37(4-6):129-281. 2. Rodil SE, Oli a es R, A za e H, Muhl S. P ope ies o ca bon ilms and hei biocompa ibili y using in- i o es s. Diamond and Rela ed Ma e ials 2003;12(3- 7):931-937. 3. Vand o co a M, Bacako a L. Adhesion, G ow h and Di e en ia ion o Os eoblas s on Su ace-Modi ied Ma e ials De eloped o Bone Implan s. Physiological Resea ch 2011;60(3):403-417. 4. Haue R, Mulle U. An o e iew on ailo ed ibological and biological beha io o diamond-like ca bon. Diamond and Rela ed Ma e ials 2003;12(2):171-177. 5. Do ne -Reisel A, Ga ne G, Reisel G, I me G. Diamond-like ca bon ilms o polye hylene emo al pa s: Raman and FT-IR spec oscopy be o e and a e incuba ion in simula ed body liquid. Analy ical and Bioanaly ical Chemis y 2008;390(6):1487-1493. 6. Ma WJ, Ruys AJ, Mason RS, Ma in PJ, Benda id A, Liu ZW, Ionescu M, Z eiqa H. DLC coa ings: E ec s o physical and chemical p ope ies on biological esponse. Bioma e ials 2007;28(9):1620-1628. 7. Lou o C, Mou a CW, Ca alho N, S uebe M, Ca alei o A. The mal s abili y in oxida i e and p o ec i e en i onmen s o a-C:H cap laye on a unc ional g adien coa ing. Diamond and Rela ed Ma e ials 2011;20(2):57-63. 8. 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Ma e ials Science and Enginee ing B-Ad anced Func ional Solid-S a e Ma e ials 2010;169(1-3):89-93. 16. Singh A, Eh eshami G, Massia S, He JP, S o e RG, Raupp G. Glial cell and ib oblas cy o oxici y s udy on plasma-deposi ed diamond-like ca bon coa ings. Bioma e ials 2003;24(28):5083-5089. Page 24 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A 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). Page 32 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 33 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 34 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 35 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 36 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 37 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 38 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 39 o 43 Jou nal o Biomedical Ma e ials Resea ch: Pa A 209x297mm (150 x 150 DPI) Page 40 o 43Jou nal o Biomedical Ma e ials Resea ch: Pa A