Relevant Aspects of Piranha Passivation in Ti6Al4V Alloy Dental Meshes

  Ci a ion: C uz, N.; Gil, J.; Punse , M.; Mane o, J.M.; Tondela, J.P.; Ve degue , P.; Apa icio, C.; Rúpe ez, E. Rele an Aspec s o Pi anha Passi a ion in Ti6Al4V Alloy Den al Meshes. Coa ings 2022,12, 154. h ps://doi.o g/ 10.3390/coa ings12020154 Academic Edi o s: Jasmina P imožiˇc and Vincenzo D’An ò Recei ed: 28 Decembe 2021 Accep ed: 24 Janua y 2022 Published: 27 Janua y 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). coa ings A icle Rele an Aspec s o Pi anha Passi a ion in Ti6Al4V Alloy Den al Meshes Nuno C uz 1, Ja ie Gil 1,2,* , Miquel Punse 3,4,5,6 , JoséMa ía Mane o 3,4,6, João Paulo Tondela 7,* , Pablo Ve degue 1, Con ado Apa icio 2and Elisa Rúpe ez 3,4,6 1Bioenginee ing Ins i u e o Technology, In e na ional Uni e si y o Ca alonia (UIC), 08195 Ba celona, Spain; [email p o ec ed] (N.C.); pablo e [email p o ec ed] (P.V.) 2School o Den is y, In e na ional Uni e si y o Ca alonia (UIC), 08195 Ba celona, Spain; [email p o ec ed] 3Bioma e ials, Biomechanics and Tissue Enginee ing G oup (BBT), Depa men o Ma e ials Science and Enginee ing, Technical Uni e si y o Ca alonia (UPC), 08019 Ba celona, Spain; [email p o ec ed] (M.P.); [email p o ec ed] (J.M.M.); elisa. [email p o ec ed] (E.R.) 4Ba celona Resea ch Cen e in Mul iscale Science and Enginee ing, Technical Uni e si y o Ca alonia (UPC), 08019 Ba celona, Spain 5 Inno a ion and Technology Cen e (CIT), Poly echnic Uni e si y o Ca alonia (UPC), 08034 Ba celona, Spain 6San Joan de Déu Resea ch Ins i u e (IRSJD), 08034 Ba celona, Spain 7CIROS om he Facul y o Medicine, Uni e si y o Coimb a, FMUC, 3004-531 Coimb a, Po ugal *Co espondence: xa ie [email p o ec ed] (J.G.); [email p o ec ed] (J.P.T.) Abs ac : Passi a ion o i anium alloy den al meshes cleans hei su ace and o ms a hin laye o p o ec i e oxide (TiO 2 ) on he su ace o he ma e ial o imp o e esis ance o co osion and p e en elease o ions o he physiological en i onmen . The mos common chemical agen o he passi a- ion p ocess o i anium meshes is hyd ochlo ic acid (HCl). In his wo k, we in oduce he use o Pi anha solu ion (H 2 SO 4 and H 2 O 2 ) as a passi a ing and bac e icidal agen o me allic den al meshes. Meshes o g ade 5 i anium alloy (Ti6Al4V) we e es ed a e di e en ea men s: as- ecei ed con ol (C ), passi a ed by HCl, and passi a ed by Pi anha solu ion. Physical-chemical cha ac e iza ion o all ea ed su aces was ca ied ou by scanning elec on mic oscopy (SEM), con ocal mic oscopy and sessile d op goniome y o assess meshes’ opog aphy, elemen al composi ion, oughness, we abili y and su ace ee ene gy, ha is, ele an p ope ies wi h po en ial e ec s o he biological esponse o he ma e ial. Mo eo e , open ci cui po en ial and po en iodynamic es s we e ca ied ou o e alua e he co osion beha io o he di e en ly- ea ed meshes unde physiological condi ions. Ion elease es s we e conduc ed using Induc i ely Coupled Plasma mass spec ome y (ICP-MS). The an ibac e ial ac i i y by p e en ion o bac e ial adhesion es s on he meshes was pe o med o wo di e en bac e ial s ains, Pseudomonas ae uginosa (G am-) and S ep ococcus sanguinis (G am+). Addi ionally, a bac e ial iabili y s udy was pe o med wi h he LIVE/DEAD es . We complemen ed he an ibac e ial s udy by coun ing cells a ached o he su ace o he meshes isualized by SEM. Ou esul s showed ha he passi a ion o i anium meshes wi h Pi anha solu ion imp o ed hei hyd ophilici y and con e ed a no ably highe bac e icidal ac i i y in compa ison wi h he meshes passi a ed wi h HCl. This unique esponse can be a ibu ed o di e ences in he ob ained nano ex- u es o he TiO 2 laye . Howe e , Pi anha solu ion ea men dec eased elec ochemical s abili y and inc eased ion elease as a esul o he po ous coa ing o med on he ea ed su aces, which can comp omise hei co osion esis ance. F amed by he limi a ions o his wo k, we conclude ha using Pi anha solu ion is a iable al e na i e me hod o passi a ing i anium den al meshes wi h bene icial an ibac e ial p ope ies ha me i s u he alida ion o i s ansla ion as a ea men applied o clinically-used meshes. Keywo ds: i anium; den al meshes; passi a ion; pi anha; co osion esis ance; ion elease; bac e ial adhesion Coa ings 2022,12, 154. h ps://doi.o g/10.3390/coa ings12020154 h ps://www.mdpi.com/jou nal/coa ings Coa ings 2022,12, 154 2 o 17 1. In oduc ion The amoun o bone is pa amoun o p edic ably achie e success and long- e m su i al o implan -suppo ed ehabili a ions. Ac ually, implan den is y has e ol ed o a p os he ically d i en implan placemen concep , meaning ha biology, biomechanics, unc ion and es he ics o he implan suppo ed ehabili a ion should be conside ed o he adequa e implan posi ion in bone. Al hough p ope amoun o bone is needed o go along wi h he es he ical and unc ional p os he ic design, a iable disc epancies in he a ailable bone a e seldom ound. This may occu because o p olonged oo h loss, auma, inju y o bone disease and esec ion, conduc ing o a ho izon al, e ical o combined bone de ec (Siebe ). Hence, se e al echniques and ma e ials o bone augmen a ion ha e been used concomi an wi h implan placemen o as a s aged app oach [1–4]. Following he biological p inciples o selec i e cell exclusion o egene a i e wound healing and guided issue egene a ion, hese we e la e p o en o be applicable o guided bone egene a ion also. Techniques in ol e placing a mechanical ba ie o p o ec he blood clo and o isola e he bony de ec om he su ounding connec i e and epi helial issue in asion. This space is needed o allow he os eoblas s o access he space in ended o bone egene a ion [5,6]. Ti anium igid sca olds we e success ully used o bone augmen a ion, e en ou side o he bone en elope. P esen ly, one mains eam di ec ion o 3D p in ing is biomedical applica ions, speci ically in c ea ing sca olds o medical implan s such as indi idualized i anium meshes o bone egene a ion [ 7 – 9 ]. In ecen yea s, he de elopmen o pe sonal- ized apid p o o yping medical de ices based on he digi al imaging and communica ions in medicine (DICOM) iles p o ided by compu e ized omog aphy/cone beam compu - e ized omog aphy (CT/CBCT) scans has deeply in ensi ied [ 10 ]. Based on he pa ien ’s bone de ec and eso ing o compu e aided design (CAD) so wa e, i is possible o design medical de ices wi h he in en o ec ea ing he los idimensional bone ana omy. Rega dless o he p oduc ion echnique o any implan able de ices, i is manda o y o con ol he cha ac e is ics such as pe meabili y, su ace opog aphy and oughness, and op imize hei biological pe o mance [ 11 – 16 ]. High deg ees o oughness ep esen a majo isk o ionic leakage om he ma e ial [ 17 ] and he bac e ial adhesion can be inc eased, wi h he consequence o implan ailu es [ 10 ]. Smoo h su aces a e able o slow down he biological p ocesses a he in e ace, keeping he i anium oxidized laye p ope ies una ec ed o longe ime pe iods [ 9 ]. The associa ed co ec mic o- and nano- oughness le el can s imula e os eoblas di e en ia ion, p oli e a ion and p oduc ion o bo h ma ix and local g ow h ac o s [ 10 ]. Fu he mo e, changes in oughness co ela e wi h selec i e p o ein adso p ion, collagen syn hesis and he ma u a ion o chond ocy es, which all signi ican ly in luence he implan ’s osseoin eg a ion [10]. I is well known ha he implan –li ing issues in e ac ions depend on he su ace p ope ies, such as oughness, we abili y, su ace ene gy and chemical composi ion, among o he s. Bioma e ials esea ch should op imize, a di e en scales, he su ace cha ac e is ics in o de o imp o e di e en unc ions: bioac i i y, osseoin eg a ion o bac e icide beha io . In addi ion, i anium meshes a e suscep ible o co osion due o he p esence o me als o di e en chemical na u e in he mou h, as well as he elease o i anium ions in o he en i onmen which mus be aken in o accoun [ 11 – 13 ]. I has been long ecognized ha he co osion p oduc s o med as a esul o me al–en i onmen in e ac ions ha e a signi ican bea ing on he biocompa ibili y and long- e m s abili y o he p os heses/implan . The ma e ial used mus no cause any biological ad e se eac ion and mus e ain i s o m and p ope ies [ 11 , 12 ] du ing unc ion. Human s oma ogna hus is subjec ed o a ying changes in pH and empe a u e owing o di e ences in local, sys emic, en i onmen al, economic and social condi ions o each indi idual. Co osion can esul om he p esence o a numbe o co osi e species such as hyd ogen ion (H + ), sul ide compounds (S 2− ), dissol ed oxygen, ee adicals (O 2− , O − ), and chlo ide ion (Cl − ) esul ing in he me al su ace b eakdown and a consequen ad e se issue eac ion [ 13 ]. In addi ion, he e ec o Coa ings 2022,12, 154 3 o 17 bac e ia can lead o he appea ance o bac e ial plaque which will a ec bone egene a ion and cause in lamma ion in he pa ien [14–16]. Passi a ion is, in gene al, an oxida ion eac ion ob ained by chemical o elec ochem- ical p ocess which p omo es he o ma ion and inc easing o he hickness o p o ec i e laye s [ 10 – 13 ]. The e ec o passi a ion and oxida i e agen s and he ole o i anium oxide as he physico-chemical cha ac e is ics o he su ace a e poo ly s udied and unde - s ood [17–20]. In i o s udies ha e implied ha he nega i ely cha ged and hyd ophilic TiO 2 laye is, in ac , he key ac o o he o e all biocompa ibili y as i egula es he p o ein ad- so p ion [ 9 ]. Fo he pa icula case o he den is y, coun less s udies ha e al eady been conduc ed in o de o gua an ee he implan a ion sa e y. Usually, no in lamma o y esponse signs a e ound in he o al issue adjacen o i anium implan s; howe e , i is impo an o no e ha o some pa ien s, hype sensi i i y can be induced [9]. In his wo k, he aim was o s udy an al e na i e passi a ion me hod using he so- called Pi anha solu ion. The Pi anha solu ion is a mix u e o sul u ic acid and hyd ogen pe oxide. We s udied he e ec s o Pi anha solu ion ea men on su ace physical-chemical p ope ies, chemical deg ada ion (co osion and elease o ions) and an imic obial ac i i y agains G am-posi i e and G am-nega i e bac e ia. 2. Ma e ials and Me hods 2.1. Samples One hund ed wen y G ade 5 i anium alloy (Ti6Al4V) meshes (BoneEasy, A ada, Po ugal) we e used. Figu e 1shows he mesh and i s applica ion as a memb ane wi h calcium phospha e. We wo ked wi h 3 g oups o samples: Con ol: as- ecei ed ma e ial. HCl passi a ion: The meshes we e imme sed in a solu ion o hyd ochlo ic acid (HCl) 20% ( ) o 40 s a oom empe a u e (HCl g oup). This is he gold-s anda d passi a ion ea men o den al implan s and p os hesis. Pi anha passi a ion: The meshes we e imme sed in a solu ion o Pi anha, which is a mix u e o sul u ic acid 96% ( ) and a 50:50 a io o hyd ochlo ic acid (HCl) 20% ( ) and hyd ogen pe oxide 30% ( ) o 2 h. Pi anha solu ions a e a mix u e o concen a ed sul u ic acid wi h hyd ogen pe oxide, usually in a a io o 3:1 o 7:1. They a e used o emo e ace amoun s o o ganic esidues, such as pho o esis , om subs a es. The mixing p ocedu e is an exo he mic eac ion ha can each empe a u es o 100 ◦ C o highe . The eac ion o hyd ogen pe oxide on concen a ed sul u ic acid p oduces highly ac i a ed and oxidizing pe oxymonosul u ic acid (H 2 SO 5 ), also called Ca o’s acid [ 1 ]. Howe e , he e a e many di e en mix u e a ios ha a e commonly used, and all a e called Pi anha. The addi ion o NH 4 OH in o de o accele a e he decomposi ion o H 2 O 2 o he addi ion o HCl, as in his esea ch, a o s cleanness and inc eases he oxide s abiliza ion. Pi anha solu ion mus be p epa ed wi h g ea ca e. I is highly co osi e and an ex emely powe ul oxidize . Su aces mus be easonably clean and comple ely ee o o ganic sol en s om p e ious washing s eps be o e coming in o con ac wi h he solu ion. Pi anha solu ion cleans by decomposing o ganic con aminan s, and a la ge amoun o con aminan will cause iolen bubbling and a elease o gas ha can cause an explosion [21]. A e ea men , all samples we e cleaned a sequence o 3 ul asonic ba hs (3 min each): wo consecu i e wi h dis illed wa e , ollowed by one wi h e hanol. Coa ings 2022,12, 154 4 o 17 Coa ings 2022, 12, x FOR PEER REVIEW 4 o 18 Figu e 1. G ade 5 i anium mesh used in his s udy. 2.2. Su ace Cha ac e iza ion Roughness o all g oups was de e mined using an Olympus LEXT OLS3100 con ocal mic oscope (Olympus, Tokyo, Japan). Th ee samples pe g oup we e es ed and 3 meas- u emen s pe sample we e aken a ×1000 magni ica ion. The pa ame e s Ra and Rz we e de e mined. Ra co esponds o he a i hme ic mean o he absolu e alues o he de ia- ions o he p o iles o a gi en leng h o he sample. Rz co esponds o he sum o he maximum peak heigh and he maximum alley dep h wi hin he sampling leng h. [21]. The wa e sessile d op echnique was used o he measu emen o he con ac angle, θ, o med be ween he wa e d op and he su ace. The g ea e he con ac angle, he lowe he we abili y and ice e sa. Fo angles less han 10°, he su ace is conside ed supe hy- d ophilic, o angles be ween 10° and 90° su aces a e hyd ophilic and o angles g ea e han 90°, su aces a e conside ed hyd ophobic. A d ople gene a ion sys em equipped wi h a 500 μL Hamil on sy inge wi h mic ome ic displacemen con ol was used o con- ol he olume (3 μL) and o deposi he d ople . The analysis was pe o med using a gonyome e wi h d op p o ile image cap u e (Con ac Angle Sys em OCA15plus, Da aPhysics, Filde s ad , Ge many) and analyzed wi h SCA20 so wa e (Da aPhysics, Filde s ad , Ge many) [22,23]. To calcula e he su ace ee ene gy, he con ac angle was measu ed wi h wo di e - en liquids, wa e and diiodome hane. The con ac angle measu emen s o diiodome hane we e ob ained ollowing he same p ocedu e used o measu e wa e con ac angles [22]. The su ace ee ene gy and i s pola (γp) and dispe si e (γd) componen s we e hen cal- cula ed using he Owens and Wend equa ion [17]: ))()((2)cos1( 2/1 p S p L 2/1d S d LL  (1) Su ace mo phology o he samples was analyzed wi h a ocused ion beam Zeiss Neon40 FE-SEM (Ca l Zeiss NTS GmbH, Obe kochen, Ge many). Images o uncoa ed samples we e aken a a wo king dis ance o 7 mm and an accele a ing ol age o 5 kV. An EDS de ec o (INCA Pen aFETx3 sys em, Ox o d Ins umen s, Abingdon, UK) was used o de ec sil e p esence on he su ace o he samples. This mic oscope has a esol - ing powe o 3 nm and allows he obse a ion o he nano ex u es p oduced by he eac- ion o he Pi anha solu ion wi h he Ti6Al4V alloy. Figu e 1. G ade 5 i anium mesh used in his s udy. 2.2. Su ace Cha ac e iza ion Roughness o all g oups was de e mined using an Olympus LEXT OLS3100 con ocal mic oscope (Olympus, Tokyo, Japan). Th ee samples pe g oup we e es ed and 3 mea- su emen s pe sample we e aken a × 1000 magni ica ion. The pa ame e s Ra and Rz we e de e mined. Ra co esponds o he a i hme ic mean o he absolu e alues o he de ia ions o he p o iles o a gi en leng h o he sample. Rz co esponds o he sum o he maximum peak heigh and he maximum alley dep h wi hin he sampling leng h [21]. The wa e sessile d op echnique was used o he measu emen o he con ac angle, θ , o med be ween he wa e d op and he su ace. The g ea e he con ac angle, he lowe he we abili y and ice e sa. Fo angles less han 10 ◦ , he su ace is conside ed supe hyd ophilic, o angles be ween 10 ◦ and 90 ◦ su aces a e hyd ophilic and o angles g ea e han 90 ◦ , su aces a e conside ed hyd ophobic. A d ople gene a ion sys em equipped wi h a 500 µ L Hamil on sy inge wi h mic ome ic displacemen con ol was used o con ol he olume (3 µ L) and o deposi he d ople . The analysis was pe o med using a gonyome e wi h d op p o ile image cap u e (Con ac Angle Sys em OCA15plus, Da aPhysics, Filde s ad , Ge many) and analyzed wi h SCA20 so wa e (Da aPhysics, Filde s ad , Ge many) [22,23]. To calcula e he su ace ee ene gy, he con ac angle was measu ed wi h wo di e en liquids, wa e and diiodome hane. The con ac angle measu emen s o diiodome hane we e ob ained ollowing he same p ocedu e used o measu e wa e con ac angles [ 22 ]. The su ace ee ene gy and i s pola ( γp ) and dispe si e ( γd ) componen s we e hen calcula ed using he Owens and Wend equa ion [17]: γL·(1+cos θ) = 2·((γd L·γd S)1/2 + (γp L·γp S)1/2)(1) Su ace mo phology o he samples was analyzed wi h a ocused ion beam Zeiss Neon40 FE-SEM (Ca l Zeiss NTS GmbH, Obe kochen, Ge many). Images o uncoa ed samples we e aken a a wo king dis ance o 7 mm and an accele a ing ol age o 5 kV. An EDS de ec o (INCA Pen aFETx3 sys em, Ox o d Ins umen s, Abingdon, UK) was used o de ec sil e p esence on he su ace o he samples. This mic oscope has a esol ing powe o 3 nm and allows he obse a ion o he nano ex u es p oduced by he eac ion o he Pi anha solu ion wi h he Ti6Al4V alloy. 2.3. Co osion Beha io A o al o 60 samples, (n= 20) o each g oup o samples, we e used o he co osion es s. The es a ea o each sample was 19.6 mm 2 . The elec oly e o all es s was Hank’s solu ion (Table 1), which is a saline luid ha closely cap u es he ion composi ion o he human se um en i onmen . Coa ings 2022,12, 154 5 o 17 Table 1. Composi ion o Hank’s solu ion. Chemical P oduc Composi ion (mM) K2HPO40.44 KCl 5.4 CaCl21.3 Na2HPO40.25 NaCl 137 NaHCO34.2 MgSO41.0 C6H12O65.5 The elec ochemical cell used was a polyp opylene (PP) con aine wi h a capaci y o 185 mL and a me hac yla e lid wi h 6 holes o he in oduc ion o he sample, he e e ence elec ode and he coun e elec ode (Figu e 2). Fo bo h he open ci cui po en ial measu emen es s and he po en iodynamic es s, he e e ence elec ode used was a calomel elec ode (sa u a ed KCl), wi h a po en ial o 0.241 V compa ed o he s anda d hyd ogen elec ode. All es s we e pe o med a oom empe a u e and in a Fa aday cage o a oid he in e ac ion o ex e nal elec ic ields. Fo he open ci cui po en ial measu emen es s, only he sample and he e e ence elec ode we e placed in he elec ochemical cell. Tes s we e ca ied ou o 5 h o all he samples, aking measu emen s e e y 10 s. The po en ial was conside ed o be s abilized when he a ia ion o he po en ial is less han 2 mV o e a pe iod o 30 min as indica ed in he ASTM G31 s anda d [23]. This es assesses which ma e ials a e mo e noble (highe po en ial) and hus, less suscep ible o co ode. The da a and he E- cu es we e ob ained using he Powe Sui e so wa e (Schneiede Elec ic, Ruil-Malmaison, F ance) wi h he Powe Co -Open ci cui (Schneiede Elec ic, Ruil-Malmaison, F ance). Coa ings 2022, 12, x FOR PEER REVIEW 5 o 18 2.3. Co osion Beha io A o al o 60 samples, (n = 20) o each g oup o samples, we e used o he co osion es s. The es a ea o each sample was 19.6 mm2. The elec oly e o all es s was Hank’s solu ion (Table 1), which is a saline luid ha closely cap u es he ion composi ion o he human se um en i onmen . Table 1. Composi ion o Hank’s solu ion. Chemical P oduc Composi ion (mM) K2HPO4 0.44 KCl 5.4 CaCl2 1.3 Na2HPO4 0.25 NaCl 137 NaHCO3 4.2 MgSO4 1.0 C6H12O6 5.5 The elec ochemical cell used was a polyp opylene (PP) con aine wi h a capaci y o 185 mL and a me hac yla e lid wi h 6 holes o he in oduc ion o he sample, he e e - ence elec ode and he coun e elec ode (Figu e 2). Fo bo h he open ci cui po en ial measu emen es s and he po en iodynamic es s, he e e ence elec ode used was a cal- omel elec ode (sa u a ed KCl), wi h a po en ial o 0.241 V compa ed o he s anda d hy- d ogen elec ode. All es s we e pe o med a oom empe a u e and in a Fa aday cage o a oid he in e ac ion o ex e nal elec ic ields. Fo he open ci cui po en ial measu emen es s, only he sample and he e e ence elec ode we e placed in he elec ochemical cell. Tes s we e ca ied ou o 5 h o all he samples, aking measu emen s e e y 10 s. The po en ial was conside ed o be s abilized when he a ia ion o he po en ial is less han 2 mV o e a pe iod o 30 min as indica ed in he ASTM G31 s anda d [23]. This es assesses which ma e ials a e mo e noble (highe po en ial) and hus, less suscep ible o co ode. The da a and he E- cu es we e ob ained using he Powe Sui e so wa e (Schneiede Elec ic, Ruil-Malmaison, F ance) wi h he Powe Co -Open ci cui (Schneiede Elec ic, Ruil-Malmaison, F ance). Figu e 2. Expe imen al se up used o assessing co osion esis ance. Cyclic po en iodynamic pola iza ion cu es we e ob ained o he 3 s udy g oups ollowing he ASTM G5 s anda d. In his es , a a iable elec ical po en ial is imposed by he po en ios a be ween he sample and he e e ence elec ode, causing a cu en o low be ween he sample and he coun e elec ode. The coun e elec ode used was pla inum [17,24–25]. Be o e s a ing he es , he sys em was allowed o s abilize by means o an Figu e 2. Expe imen al se up used o assessing co osion esis ance. Cyclic po en iodynamic pola iza ion cu es we e ob ained o he 3 s udy g oups ollowing he ASTM G5 s anda d. In his es , a a iable elec ical po en ial is imposed by he po en ios a be ween he sample and he e e ence elec ode, causing a cu en o low be ween he sample and he coun e elec ode. The coun e elec ode used was pla inum [17,24,25]. Be o e s a ing he es , he sys em was allowed o s abilize by means o an open ci cui es o 1 h. A e s abiliza ion, he po en iodynamic es was launched, pe o ming a cyclic sweep om − 0.8 mV o 1.7 mV a a speed o 2 mV/s. These pa ame- e s we e en e ed in o he Powe Sui e p og am using he Powe Co -Cyclic Pola iza ion unc ion o ob ain he cu es. The pa ame e s s udied we e: 1. ico (µA/cm2)—co osion cu en densi y; 2. E co (mV)—co osion po en ial: alue a which he cu en densi y changes om ca hodic o anodic; 3. E ep (mV)— epassi a ion po en ial: po en ial a which he passi e laye egene a es; 4. Ep(mV)—pi ing po en ial: alue a which pi ing co osion may occu ; Coa ings 2022,12, 154 6 o 17 5. ip(µA/cm2)—passi a ion cu en densi y; 6. ip (µA/cm2)— epassi a ion cu en densi y. The E co and i co pa ame e s a e ob ained by ex apola ing he Ta el slopes. The Ta el slopes a e also used o ob ain he Ta el coe icien s: anodic ( β a) and ca hodic ( β c). These coe icien s ep esen he slopes o he anodic and ca hodic b anch, espec i ely. In acco dance wi h he ASTM G102-89 s anda d [ 23 – 26 ], hese alues a e hen used o calcula e he pola iza ion esis ance (R p ) using he S e n–Gea y exp ession and he co osion a e (CR in mm/yea ) [24–28]. Rp =βa·βc 2.303 ·(βa+βc)·ico (2) The pola iza ion esis ance indica es he esis ance o he sample o co osion when subjec ed o small a ia ions in po en ial. A o al o 30 po en iodynamic es s we e ca ied ou , ob aining a leas 10 cu es pe g oup. CR =K1·ico ρ·EW (3) 2.4. Ion Release Fi e samples om each g oup we e used o he me al ion eco e y es . A e weigh- ing he samples (m = 0.206 g) and ollowing he ISO 10993-12 s anda d [ 26 ], a weigh adjus men was made a he a e o 1 mL o Hank’s solu ion o each 0.2 g o sample, as indica ed in he s anda d. The 5 samples o each g oup we e placed in he same Eppendo wi h 5 mL o Hank’s solu ion and s o ed a 37 ◦ C. Hank’s solu ion should be ex ac ed and s o ed in he e ige a o a e 1, 3, 7, 14 and 21 days. A e each ex ac ion, 5 mL o esh Hank’s solu ion has been eplenished in o he Eppendo con aining he samples. All Eppendo ubes should be cleaned wi h 2% Ni ic Acid and d ied be o e use. A e 21 days, he concen a ion o eleased i anium ions was measu ed, a he es imes indica ed abo e, by induc i ely coupled plasma mass spec ome y (ICP-MS) wi h he Agilen Technologies 7800 ICP-MS. 2.5. Bac e ia Analysis Two ypes o bac e ia, P. ae uginosa (Colección española de cul i os ipo, CECT 110, Valencia, Spain) and S. sanguinis (Cul u e Collec ion Uni e si y o Go henbu g, CCUG 15915, Go henbu g, Sweden), a G am-nega i e and a G am-posi i e s ain, espec i ely, we e used o he bac e ial adhesion es . Th ee samples pe g oup and bac e ial s ain we e es ed. The cul u e media and ma e ial (PBS) we e p e iously s e ilized by au ocla ing a 121 ◦C o 30 min. P io o he adhesion es , he samples we e also s e ilized. Fo his pu - pose, h ee 5 min e hanol washes we e ca ied ou in s e ile cul u e pla es. A e emo ing he e hanol, he samples we e exposed o ul a iole ligh o ano he 30 min [29,30]. The aga pla es we e cul u ed a 37 ◦ C o 24 h. F om his cul u e, he liquid inoculum was p epa ed by suspending he bac e ia in 5 mL o BHI (B ain Hea In usion) and incuba ed o 24 h a 37 ◦ C. The medium was hen dilu ed o an op ical densi y o 0.1 a a wa eleng h o 600 nm (OD600 = 0.1). Fo bac e ial adhesion, enough solu ion wi h a concen a ion equi alen o OD600 = 0.1 o co e he su aces (500 µ L/sample) was in oduced in o he well o he cul u e pla e o each sample and incuba ed a 37 ◦ C o 1 h. A e his ime, he samples we e insed wi h PBS o 5 min wice and he bac e ia we e ixed wi h a 2.5% glu a aldehyde solu ion in PBS (30 min in he e ige a o ). The glu a aldehyde solu ion was hen emo ed and he samples we e insed wi h PBS 3 imes o 5 min. Fo iabili y analysis by con ocal mic oscopy, he LIVE/DEAD BacLigh bac e ial iabili y ki (The mo Fishe , Mad id, Spain) was used [ 13 , 14 ]. A solu ion was p epa ed wi h 1.5 µ L o p opidium in 1 mL o PBS. Using a mic opipe e, a d op o his solu ion (app oxima ely 50 µ L/sample) was deposi ed on he s udy su ace and a e incuba ion a oom empe a u e in he da k o 15 min, he samples we e insed 3 imes wi h PBS o Coa ings 2022,12, 154 7 o 17 5 min. The su aces we e hen obse ed unde a con ocal mic oscope. Th ee images pe sample we e aken a 630 × magni ica ion ( × 63 objec i e). Wa eleng hs o 488 and 561 nm we e used o de ec bac e ia wi h non-comp omised memb anes (LIVE) and comp omised memb anes (DEAD), espec i ely. P io o he obse a ion o he samples by scanning elec on mic oscopy (SEM), he samples we e dehyd a ed. Fo he dehyd a ion p ocess and he c i ical poin d ying, 10 min washes we e ca ied ou wi h e hanol solu ions o g adual concen a ions o 30%, 50%, 70%, 80%, 90%, 95% and 100%. They we e hen le o d y o 24 h a oom empe a u e. Then, samples we e coa ed wi h pla inum o 5 s be o e obse a ion unde he mic oscope. Ten images o each sample we e aken a 20,000 × magni ica ions o bac e ial quan i ica ion on each su ace. 2.6. S a is ical Analysis All esul s we e exp essed as mean and s anda d de ia ion excep o he bac e ial adhesion es esul s which we e exp essed as median and s anda d e o . The compa a i e T.TEST (wi h he Excel so wa e) was ca ied ou be ween he di e en g oups a 95%, which means ha o alues o p< 0.05, he e a e signi ican di e ences. 3. Resul s Figu e 3shows SEM images o he su aces o he i anium alloy a e passi a ion ea men s. No signi ican a ia ions be ween he con ol and HCl ea men we e de ec ed and bo h ypes o su aces clea ly showed machining ma ks. Machining ma ks in HCl- passi a ed su aces we e ligh e han in as-machined su aces, p obably due o he e ec o he highe concen a ion o he acid. Howe e , on he su ace o he samples subjec ed o he Pi anha passi a ion ea men , he acid a ack almos comple ely emo ed he machining ma ks and, no ably, p oduced a homogenously-dis ibu ed and commonly-ob ained su ace nano ex u e in he o m o nanoca i ies (Figu e 4) [15]. Coa ings 2022, 12, x FOR PEER REVIEW 8 o 18 Figu e 3. (A) Su aces o g ade 5 Ti alloy ea ed wi h di e en passi a ion me hods; (B) a highe magni ica ions. Figu e 4. Nano ex u e o i anium alloy a e Pi anha passi a ion ea men obse ed by high- eso- lu ion scanning elec on mic oscopy. The di e en passi a ion ea men s on he i anium alloy meshes, ei he wi h HCl o Pi anha solu ion, did no al e he a e age oughness (Ra), as no s a is ically signi ican di e ences we e obse ed wi h espec o he con ol g oup (Table 2). Howe e , he Pi a- nha ea men showed s a is ically signi ican lowe Rz alues wi h espec o he o he g oups. These esul s sugges ha he Pi anha solu ion ea men a acked he i anium, educing machining ailu es and c ea ing an oxide laye ha educes he di e ences be- ween alleys and peaks. The la ge di e ence be ween he Ra and Rz alues shows ha we ha e wo ypes o ex u e (Figu e 5), one associa ed wi h he u ning ma ks esponsible o he high Rz alues and he o he he nano ex u e associa ed wi h he passi a ion ea - men . Figu e 3. ( A ) Su aces o g ade 5 Ti alloy ea ed wi h di e en passi a ion me hods; ( B ) a highe magni ica ions. Coa ings 2022,12, 154 8 o 17 Coa ings 2022, 12, x FOR PEER REVIEW 8 o 18 Figu e 3. (A) Su aces o g ade 5 Ti alloy ea ed wi h di e en passi a ion me hods; (B) a highe magni ica ions. Figu e 4. Nano ex u e o i anium alloy a e Pi anha passi a ion ea men obse ed by high- eso- lu ion scanning elec on mic oscopy. The di e en passi a ion ea men s on he i anium alloy meshes, ei he wi h HCl o Pi anha solu ion, did no al e he a e age oughness (Ra), as no s a is ically signi ican di e ences we e obse ed wi h espec o he con ol g oup (Table 2). Howe e , he Pi a- nha ea men showed s a is ically signi ican lowe Rz alues wi h espec o he o he g oups. These esul s sugges ha he Pi anha solu ion ea men a acked he i anium, educing machining ailu es and c ea ing an oxide laye ha educes he di e ences be- ween alleys and peaks. The la ge di e ence be ween he Ra and Rz alues shows ha we ha e wo ypes o ex u e (Figu e 5), one associa ed wi h he u ning ma ks esponsible o he high Rz alues and he o he he nano ex u e associa ed wi h he passi a ion ea - men . Figu e 4. Nano ex u e o i anium alloy a e Pi anha passi a ion ea men obse ed by high- esolu ion scanning elec on mic oscopy. The di e en passi a ion ea men s on he i anium alloy meshes, ei he wi h HCl o Pi anha solu ion, did no al e he a e age oughness (Ra), as no s a is ically signi ican di e ences we e obse ed wi h espec o he con ol g oup (Table 2). Howe e , he Pi anha ea men showed s a is ically signi ican lowe R z alues wi h espec o he o he g oups. These esul s sugges ha he Pi anha solu ion ea men a acked he i anium, educing machining ailu es and c ea ing an oxide laye ha educes he di e ences be ween alleys and peaks. The la ge di e ence be ween he R a and R z alues shows ha we ha e wo ypes o ex u e (Figu e 5), one associa ed wi h he u ning ma ks esponsible o he high Rz alues and he o he he nano ex u e associa ed wi h he passi a ion ea men . Table 2. Roughness alues, R a and R z, o i anium alloy su aces wi h di e en passi a ion ea - men s. Di e en le e s in he same column deno e s a is ically signi ican di e ences (p< 0.05) be ween g oups. Mesh Ra (µm) Rz (µm) Con ol 0.12 ±0.03 (a) 4.95 ±0.76 (A) HCl 0.14 ±0.08 (a) 4.87 ±0.90 (A) Pi anha 0.12 ±0.05 (a) 1.90 ±0.73 (B) Coa ings 2022, 12, x FOR PEER REVIEW 9 o 18 Table 2. Roughness alues, Ra and Rz, o i anium alloy su aces wi h di e en passi a ion ea - men s. Di e en le e s in he same column deno e s a is ically signi ican di e ences (p < 0.05) be- ween g oups. Mesh Ra (μm) Rz (μm) Con ol 0.12 ± 0.03 (a) 4.95 ± 0.76 (A) HCl 0.14 ± 0.08 (a) 4.87 ± 0.90 (A) Pi anha 0.12 ± 0.05 (a) 1.90 ± 0.73 (B) Figu e 5. Roughness pa ame e s quan i ied wi h di e en passi a ion condi ions: (a) Ra and (b) Rz. We abili y, i.e., hyd ophilic/hyd ophobic cha ac e o he es ed su aces, was de e - mined measu ing he wa e con ac angle wi h he sessile d op echnique (Table 3). Fi s ly, as- ecei ed con ol su aces we e hyd ophobic wi h a con ac angle highe han 90°. Sec- ondly, all passi a ed su aces had signi ican ly highe hyd ophilici y han un ea ed con- ol su aces. Thi dly, he su aces passi a ed wi h Pi anha solu ion p oduced a signi i- can ly highe hyd ophilic ma e ial han he su aces ea ed wi h HCl. Wa e con ac an- gle, as well as pola and dispe si e componen s o SFE, a e plo ed in Figu e 6. Co esponding wi h he esul s o he we abili y o he di e en su aces, he pola componen o he su ace ee ene gy in he i anium alloy passi a ed wi h Pi anha solu- ion was he highes among all es ed su aces. The di e ences in he dispe si e and pola componen s o he su ace ee ene gy o all es ed su aces we e s a is ically signi ican [31–35]. I is widely accep ed ha inc easing he pola componen o a ma e ial’s su ace en- e gy p omo es ini ial adhesion and cell p oli e a ion [17]. Table 3. Con ac angles and componen s o he su ace ee ene gy o he di e en ly passi a ed meshes. Mesh Θ Wa e (°) Θ Diidome hane (°) γd (mJ/m2) γp (mJ/m2) SFE (mJ/m2) Con ol 102.76 ± 7.00 48.40 ± 2.32 35.15 ± 1.28 0.12 ± 0.10 35.28 ± 1.35 HCl 86.37 ± 4.12 53.54 ± 0.92 32.39 ± 0.52 3.31 ± 1.28 35.70 ± 1.60 Pi anha 49.05 ± 7.67 34.12 ± 3.94 42.37 ± 1.79 16.52 ± 4.22 58.90 ± 4.11 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 Con ol HCl Pi anha Ra (μm) a) 0 1 2 3 4 5 6 7 Con ol HCl Pi anha Rz (μm) b) Figu e 5. Roughness pa ame e s quan i ied wi h di e en passi a ion condi ions: ( a ) Ra and ( b ) Rz. Coa ings 2022,12, 154 9 o 17 We abili y, i.e., hyd ophilic/hyd ophobic cha ac e o he es ed su aces, was de e - mined measu ing he wa e con ac angle wi h he sessile d op echnique (Table 3). Fi s ly, as- ecei ed con ol su aces we e hyd ophobic wi h a con ac angle highe han 90 ◦ . Sec- ondly, all passi a ed su aces had signi ican ly highe hyd ophilici y han un ea ed con ol su aces. Thi dly, he su aces passi a ed wi h Pi anha solu ion p oduced a signi ican ly highe hyd ophilic ma e ial han he su aces ea ed wi h HCl. Wa e con ac angle, as well as pola and dispe si e componen s o SFE, a e plo ed in Figu e 6. Co esponding wi h he esul s o he we abili y o he di e en su aces, he po- la componen o he su ace ee ene gy in he i anium alloy passi a ed wi h Pi anha solu ion was he highes among all es ed su aces. The di e ences in he dispe si e and pola componen s o he su ace ee ene gy o all es ed su aces we e s a is ically signi ican [31–35]. I is widely accep ed ha inc easing he pola componen o a ma e ial’s su ace ene gy p omo es ini ial adhesion and cell p oli e a ion [17]. Table 3. Con ac angles and componen s o he su ace ee ene gy o he di e en ly passi a ed meshes. Mesh ΘWa e (◦) ΘDiidome hane (◦) γd (mJ/m2) γp (mJ/m2) SFE (mJ/m2) Con ol 102.76 ±7.00 48.40 ±2.32 35.15 ±1.28 0.12 ±0.10 35.28 ±1.35 HCl 86.37 ±4.12 53.54 ±0.92 32.39 ±0.52 3.31 ±1.28 35.70 ±1.60 Pi anha 49.05 ±7.67 34.12 ±3.94 42.37 ±1.79 16.52 ±4.22 58.90 ±4.11 Coa ings 2022, 12, x FOR PEER REVIEW 10 o 18 Figu e 6. θ alues (a) and SFE alues (b) o cpTi ea ed wi h di e en passi a ion condi ions. Table 4 shows ha he highes open ci cui co osion po en ial alues (EOCP) we e ob ained o i anium alloy su aces ea ed wi h HCl. The e o e, HCl passi a ion p o- duces he su aces wi h he leas endency o co osion, and he e o e he bes co osion beha io . Con e sely, su aces ea ed wi h he Pi ahna solu ion showed he lowes al- ues in open ci cui , which indica ed he highes endency o co osion. The po en iody- namic s udies con i med ha he ea men ha p oduced su aces wi h he bes co osion esis ance was using HCl, as hese passi a ed su aces showed he lowes alues o co o- sion cu en densi y (ico ) and co osion a e (Vc). In addi ion, he HCl- ea ed samples show he highes esis ance o pola iza ion (Rp). The Pi anha solu ion should p oduce he hickes p o ec i e TiO2 laye ; howe e , su aces passi a ed wi h Pi anha did no ha e an imp o ed co osion beha io wi h espec o he con ol samples. Mo eo e , only in sam- ples ea ed wi h Pi anha solu ion pi ing co osion could be obse ed a e he po en- iodynamic es s (Figu e 7). Table 4. Elec ochemical and co osion pa ame e s assessed o Ti alloy meshes wi h di e en pas- si a ion ea men s. Mesh EOCP (mV) ico (μA/cm2) Rp (MΩ/cm2) Eco (V) Vc (μm/yea ) Con ol −196 ± 01 0.027 ± 0.008 2.428 ± 0.390 −361 ± 14 0.233 ± 0.066 HCl −145 ± 11 0.018 ± 0.005 2.479 ± 0.083 −536 ± 39 0.176 ± 0.048 Pi anha −206 ± 27 0.056 ± 0.006 1.102 ± 0.149 −447 ± 26 0.488 ±0.047 0 20 40 60 80 100 120 Con ol HCl Pi anha θ, (o) a) 0 10 20 30 40 50 60 70 Con ol HCl Pi anha SFE To al (𝒎𝑱/𝒎𝟐) ϒsd ϒsp b) Figu e 6. θ alues (a) and SFE alues (b) o cpTi ea ed wi h di e en passi a ion condi ions. Table 4shows ha he highes open ci cui co osion po en ial alues (E OCP ) we e ob ained o i anium alloy su aces ea ed wi h HCl. The e o e, HCl passi a ion p oduces he su aces wi h he leas endency o co osion, and he e o e he bes co osion beha io . Con e sely, su aces ea ed wi h he Pi ahna solu ion showed he lowes alues in open ci cui , which indica ed he highes endency o co osion. The po en iodynamic s udies con i med ha he ea men ha p oduced su aces wi h he bes co osion esis ance was using HCl, as hese passi a ed su aces showed he lowes alues o co osion cu en densi y (i co ) and co osion a e (V c ). In addi ion, he HCl- ea ed samples show he highes esis ance o pola iza ion (R p ). The Pi anha solu ion should p oduce he hickes p o ec i e TiO 2 laye ; howe e , su aces passi a ed wi h Pi anha did no ha e an imp o ed co osion beha io wi h espec o he con ol samples. Mo eo e , only in samples ea ed wi h Pi anha solu ion pi ing co osion could be obse ed a e he po en iodynamic es s (Figu e 7). Coa ings 2022,12, 154 16 o 17 19. Va iola, F.; F ancis-Zalzal, S.; Leduc, A.; Ba beau, J.; Nanci, A. Oxida i e nanopa e ning o i anium gene a es mesopo ous su aces wi h an imic obial p ope ies. In . J. Nanomed. 2014,9, 2319–2325. [C ossRe ] 20. B une e, D.M.; Cheh oudi, B. The e ec s o he su ace opog aphy o mic omachined i anium subs a a on cell beha io in i o and in i o. J. 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