Bacteriostatic Poly Ethylene Glycol Plasma Coatings for Orthodontic Titanium Mini-Implants

Ci a ion: Rod iguez-Fe nandez, J.C.; Pas o , F.; Ba e a Mo a, J.M.; B izuela, A.; Puigdolle s, A.; Espina , E.; Gil, F.J. Bac e ios a ic Poly E hylene Glycol Plasma Coa ings o O hodon ic Ti anium Mini-Implan s. Ma e ials 2022,15, 7487. h ps:// doi.o g/10.3390/ma15217487 Academic Edi o s: Ma ia F ancesca S ond ini and And ea Sc iban e Recei ed: 21 Sep embe 2022 Accep ed: 23 Oc obe 2022 Published: 25 Oc obe 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/). ma e ials A icle Bac e ios a ic Poly E hylene Glycol Plasma Coa ings o O hodon ic Ti anium Mini-Implan s Juan Ca los Rod iguez-Fe nandez 1, F ancisco Pas o 1, Jose Ma ia Ba e a Mo a 1, A i za B izuela 2, And eu Puigdolle s 3,*, Edua do Espina 1and F. Ja ie Gil 4,* 1Dep . O odoncia, Facul ad de Odon ología, Uni e sidad de Se illa, A icena s/n, 41009 Se illa, Spain 2Facul ad de Odon ología, Uni e sidad Eu opea Miguel de Ce an es, C/del Pad e Julio Che alie 2, 47012 Valladolid, Spain 3Dep . O odoncia, Facul ad de Odon ología, Uni e sidad In e nacional de Ca alunya, Josep T ue a s/n, San Cuga del Vallés, 08195 Ba celona, Spain 4 Bioenginee ing Ins i u e o Technology, Facul ad de Medicia y Ciencias de la Salud, Uni e sidad In e nacional de Ca alunya, Josep T ue a s/n, San Cuga del Vallés, 08195 Ba celona, Spain *Co espondence: [email p o ec ed] (A.P.); xa ie [email p o ec ed] (F.J.G.) Abs ac : Ti anium mini-implan s a e used as ancho age o o hodon ic oo h mo emen s. Howe e , hese implan s p esen p oblems due o he in ec ion o su ounding issues. The aim o his wo k was o ob ain a polye hylene glycol (PEG) laye by plasma in o de o achie e a bac e ios a ic su ace. Ti anium su aces we e ac i a ed by a gon plasma and, a e , by PEG plasma wi h di e en powe s (100, 150 and 200 W) o 30 and 60 min. The oughness was de e mined by whi e ligh in e e ome e mic oscopy and he we abili y was de e mined by he con ac angle echnique. Su ace chemical composi ions we e cha ac e ized by X- ay pho oelec on spec oscopy (XPS) and cy ocompa ibili y and cell adhesion s udies we e pe o med wi h ib oblas (hFFs) and os eoblas (SAOS-2) cells. Bac e ial cul u es wi h Spec ococcus Sanguinis and Lac obacillus Sali a ius we e pe o med, and bac e ial coloniza ion was de e mined. The esul s showed ha plasma ea men s do no a ec he oughness. Plasma makes he su aces mo e hyd ophilic by dec easing he con ac angles om 64.2 ◦ o i anium o 5.2 ◦ o a gon-ac i a ed i anium, wi h alues anging om 12 ◦ o 25 ◦ o he di e en PEG ea men s. The plasma has wo e ec s: he cleaning o he su ace and he o ma ion o he PEG laye . The biocompa ibili y esul s we e, o all cases, highe han 80%. The polyme iza ion ea men wi h PEG educed he adhesion o hFFs om 7000 o 6000 and, o SAOS-2, om 14,000 o 6500, o pu e i anium and hose ea ed wi h PEG, espec i ely. Bac e ial adhesion was also educed om 600 o 300 CFU/mm 2 o Spe ococcuns Sanguinis and om 10,000 o 900 CFU/mm 2 o Lac obacillus Sali a ius. The bes bac e ios a ic ea men co esponded o PEG a 100 W and 30 s. As a consequence, he PEG coa ing would signi ican ly p e en he o ma ion o bac e ial bio ilm on he su ace o i anium mini-implan s. Keywo ds: bac e ios a ic beha io ; mini-implan s; poly e hylene glycol; i anium; o hodon ics 1. In oduc ion O hodon ic ea men equi es a balance in he o hodon ic biomechanics. Ancho age con ol plays he main ole o he o hodon ic o ces. This ancho age con ol is undamen al o success ul o hodon ic ea men . Se e al echniques ha ein o ce ancho age ha e been used in o hodon ic he apies o mul i-b acke o o aes he ic aligne s. Fo bo h, addi ional ancho age suppo s a e o en needed o suppo he ancho ing ee h, whe eas in ao al aids o ein o ce he ancho age a e well-accep ed due o he high loads, and ex ao al sys ems ha lack com o , such as headgea , a e o en abandoned by pa ien s. Ti anium den al implan s, because o hei excellen capaci y in osseoin eg a ion, p o ide an impo an igid s abili y o bone–implan ancho age and se e as he bes in ao al ancho age de ices [1–3] . Since he las decade o he las cen u y, unlike he osseoin eg a ed Ma e ials 2022,15, 7487. h ps://doi.o g/10.3390/ma15217487 h ps://www.mdpi.com/jou nal/ma e ials Ma e ials 2022,15, 7487 2 o 13 den al implan s, emo able i anium mini-implan s ha e been ex ensi ely used o p o ide excellen bone ancho age ha esis s high-o hodon ic o ces. These mini-implan s a e easy o inse and emo e. Mini-implan s, which we e o iginally simply su gical minisc ews, ha e been de eloped and op imized o apply o many o hodon ic s a egies. Mos im- po an ly, gi en hei small size, hey can be placed in he al eola bone o adjacen ee h wi hou damaging oo s [ 1 – 4 ]. The applica ions o mini-implan s in o hodon ics a e highly e sa ile as can be seen in Figu e 1. Ma e ials 2022, 15, 7487 2 o 14 abandoned by pa ien s. Ti anium den al implan s, because o hei excellen capaci y in osseoin eg a ion, p o ide an impo an igid s abili y o bone–implan ancho age and se e as he bes in ao al ancho age de ices [1–3]. Since he las decade o he las cen u y, unlike he osseoin eg a ed den al implan s, emo able i anium mini-implan s ha e been ex ensi ely used o p o ide excellen bone ancho age ha esis s high-o hodon ic o ces. These mini-implan s a e easy o inse and emo e. Mini-implan s, which we e o iginally simply su gical minisc ews, ha e been de eloped and op imized o apply o many o hodon ic s a egies. Mos impo an ly, gi en hei small size, hey can be placed in he al eola bone o adjacen ee h wi hou damaging oo s [1–4]. The applica ions o mini- implan s in o hodon ics a e highly e sa ile as can be seen in Figu e 1. (A) (B) Figu e 1. Mini-implan s used in o hodon ic he apies. (A) As ancho age o ee h mo emen . (B) To suppo a maxilla y expande [5]. Howe e , he bigges p oblem encoun e ed in he den al clinic is he bac e ial coloniza ion ha can be c ea ed on he i anium su ace. Bio ilm causes gingi al in lamma ion and bone loss and signi ican ly educes he a achmen o he bone. In addi ion, in lamma ion o he gingi al issue a ound he head o he mini-implan is a isk ac o o i s s abili y [6–8]. In Figu e 2, i anium mini-implan s colonized by bac e ia can be obse ed. In ec ions ela ed o i anium mini-implan s a e di icul o ea since bac e ial adhesion o en leads o he o ma ion o a bio ilm, which is a mul i-species communi y embedded in a polysaccha ide ex acellula ma ix p oduced by he bac e ia. The bio ilm p o ec s he bac e ia communi y agains he immune esponse and p o ides hem wi h esis ance o an ibio ic ea men s [7–9]. Figu e 1. Mini-implan s used in o hodon ic he apies. ( A ) As ancho age o ee h mo emen . ( B ) To suppo a maxilla y expande [5]. Howe e , he bigges p oblem encoun e ed in he den al clinic is he bac e ial colo- niza ion ha can be c ea ed on he i anium su ace. Bio ilm causes gingi al in lamma ion and bone loss and signi ican ly educes he a achmen o he bone. In addi ion, in lam- ma ion o he gingi al issue a ound he head o he mini-implan is a isk ac o o i s s abili y [ 6 – 8 ]. In Figu e 2, i anium mini-implan s colonized by bac e ia can be obse ed. In ec ions ela ed o i anium mini-implan s a e di icul o ea since bac e ial adhesion o en leads o he o ma ion o a bio ilm, which is a mul i-species communi y embedded in a polysaccha ide ex acellula ma ix p oduced by he bac e ia. The bio ilm p o ec s he bac e ia communi y agains he immune esponse and p o ides hem wi h esis ance o an ibio ic ea men s [7–9]. One o he bes known an i ouling polyme s is poly (e hylene glycol) (PEG) [ 10 – 12 ]. PEG molecula chains a e belie ed o esis p o ein adso p ion by wo mechanisms: s e ic epulsion due o he su ace ension o he PEG when adso bed by he i anium subs a e and he ba ie ac ion c ea ed by he s uc u ed wa e associa ed wi h he PEG [ 13 , 14 ]. Many di e en app oaches ha e been used o immobilize PEG on he bioma e ials su ace: sel -assembly, physiso p ion, silaniza ion, elec opolyme iza ion o plasma polyme iza ion, among o he s [ 15 – 21 ]. Howe e , hough plasma polyme iza ion has been ex ensi ely used Ma e ials 2022,15, 7487 3 o 13 on polyme ic su aces [ 21 , 22 ] o ob ain PEG-like coa ings, o he bes o ou knowledge, i has no been used on i anium su aces. Ma e ials 2022, 15, 7487 3 o 14 Figu e 2. Gingi al in lamma ion a ound he head o i anium o hodon ic mini-implan s [5]. One o he bes known an i ouling polyme s is poly (e hylene glycol) (PEG) [10–12]. PEG molecula chains a e belie ed o esis p o ein adso p ion by wo mechanisms: s e ic epulsion due o he su ace ension o he PEG when adso bed by he i anium subs a e and he ba ie ac ion c ea ed by he s uc u ed wa e associa ed wi h he PEG [13,14]. Many di e en app oaches ha e been used o immobilize PEG on he bioma e ials su ace: sel -assembly, physiso p ion, silaniza ion, elec opolyme iza ion o plasma polyme iza ion, among o he s [15–21]. Howe e , hough plasma polyme iza ion has been ex ensi ely used on polyme ic su aces [21,22] o ob ain PEG-like coa ings, o he bes o ou knowledge, i has no been used on i anium su aces. In addi ion o PEG ea men s, one o he mos p omising solu ions used o induce he bac e icidal cha ac e o i anium has been TiO2 nano ube o ma ion ea men s. An ibio ics and o he d ugs can be inco po a ed in o hese nano ubes o aid osseoin eg a ion and inhibi bac e ial coloniza ion [23,24]. In addi ion, in silico s udies would be desi able o de e mine he in luence o he mini-implan designs [25–27] and o de e mine he bene i o inc easing he mechanical p ope ies o he mini-implan wi h he Ti6Al4V alloy [28]. In his wo k, PEG coa ings ha e been applied o i anium mini-implan s o o hodon ics. This con ibu ion con i ms he possibili y o ha ing bac e ios a ic coa ings on o hodon ic mini implan s. A p esen , hese mini implan s can su e bac e ial coloniza ion, leading o o hodon ic ancho age ailu e. Achie ing a coa ing ha inhibi s he o ma ion o bio ilm is o g ea clinical in e es . In his esea ch, we in end o bo h s udy he possibili y o ob aining a PEG coa ing and cha ac e ize i s opog aphical and we abili y p ope ies, as well as he cellula and mic obiological esponse, o de e mine i i can be a p omising ea men o o hodon ic mini-implan s. 2. Ma e ials and Me hods Eigh y cp- i anium mini-implan s o g ade 3 (HDC® ·M, Mineapolis, MN, USA) ha we e 2 mm in diame e and 9 mm in leng h we e used o su ace cha ac e iza ion and biological and mic obiological s udies (Figu e 3). Figu e 2. Gingi al in lamma ion a ound he head o i anium o hodon ic mini-implan s [5]. In addi ion o PEG ea men s, one o he mos p omising solu ions used o induce he bac e icidal cha ac e o i anium has been TiO 2 nano ube o ma ion ea men s. An ibio ics and o he d ugs can be inco po a ed in o hese nano ubes o aid osseoin eg a ion and inhibi bac e ial coloniza ion [ 23 , 24 ]. In addi ion, in silico s udies would be desi able o de e mine he in luence o he mini-implan designs [ 25 – 27 ] and o de e mine he bene i o inc easing he mechanical p ope ies o he mini-implan wi h he Ti6Al4V alloy [28]. In his wo k, PEG coa ings ha e been applied o i anium mini-implan s o o hodon- ics. This con ibu ion con i ms he possibili y o ha ing bac e ios a ic coa ings on o - hodon ic mini implan s. A p esen , hese mini implan s can su e bac e ial coloniza ion, leading o o hodon ic ancho age ailu e. Achie ing a coa ing ha inhibi s he o ma ion o bio ilm is o g ea clinical in e es . In his esea ch, we in end o bo h s udy he possibili y o ob aining a PEG coa ing and cha ac e ize i s opog aphical and we abili y p ope ies, as well as he cellula and mic obiological esponse, o de e mine i i can be a p omising ea men o o hodon ic mini-implan s. 2. Ma e ials and Me hods Eigh y cp- i anium mini-implan s o g ade 3 (HDC ®· M, Mineapolis, MN, USA) ha we e 2 mm in diame e and 9 mm in leng h we e used o su ace cha ac e iza ion and biological and mic obiological s udies (Figu e 3). Ma e ials 2022, 15, 7487 4 o 14 Figu e 3. O hodon ic mini-implan used in his s udy. In Figu e 4, we can see a scheme o he esea ch ca ied ou wi h he i anium mini- implan s, he p epa a ion o he samples coa ed wi h PEG plasma polyme iza ion and he cha ac e iza ion ca ied ou . Figu e 4. Schema ic ep esen a ion o he me hodology used. The me hodology o plasma ac i a ion p ocess o Ti su aces was ca ied ou as explained by Buxade a e al. [10]. These samples (n = 30) we e ea ed by adio equency low-p essu e plasma appa a us (Plasma Sys em Fem o, Diene , Ge many) using 13.52 Hz o 10 min ea men , wi h a gon a a p essu e o 0.40 mba (Figu e 5). This ea men was ealized o ac i a e he i anium su ace and, o his eason, was ca ied ou by a gon nonpolyme izing gas. Plasma polyme iza ion was pe o med igh a e plasma ac i a ion in he same eac o wi hou b eaking he acuum. The polyme iza ion p ecu so was e a (e hylene glycol) dime hyl e he ( e aglyme, Sigma Ald ich, San Louis, MO, USA) in oduced by bubbling a gon in he eac o , and he pa ame e s used we e 100 W, 0.40 mba and 1 h, acco ding o a p e ious wo k [21]. The p ocess was pe o med in pulsed mode wi h on = 20 µs and o = 20 ms. In Table 1, he di e en condi ions o he plasma polyme iza ion can be obse ed. Fi e samples we e used o each ea men (powe peak and ime). Figu e 3. O hodon ic mini-implan used in his s udy. Ma e ials 2022,15, 7487 4 o 13 In Figu e 4, we can see a scheme o he esea ch ca ied ou wi h he i anium mini- implan s, he p epa a ion o he samples coa ed wi h PEG plasma polyme iza ion and he cha ac e iza ion ca ied ou . Ma e ials 2022, 15, 7487 4 o 14 Figu e 3. O hodon ic mini-implan used in his s udy. In Figu e 4, we can see a scheme o he esea ch ca ied ou wi h he i anium mini- implan s, he p epa a ion o he samples coa ed wi h PEG plasma polyme iza ion and he cha ac e iza ion ca ied ou . Figu e 4. Schema ic ep esen a ion o he me hodology used. The me hodology o plasma ac i a ion p ocess o Ti su aces was ca ied ou as explained by Buxade a e al. [10]. These samples (n = 30) we e ea ed by adio equency low-p essu e plasma appa a us (Plasma Sys em Fem o, Diene , Ge many) using 13.52 Hz o 10 min ea men , wi h a gon a a p essu e o 0.40 mba (Figu e 5). This ea men was ealized o ac i a e he i anium su ace and, o his eason, was ca ied ou by a gon nonpolyme izing gas. Plasma polyme iza ion was pe o med igh a e plasma ac i a ion in he same eac o wi hou b eaking he acuum. The polyme iza ion p ecu so was e a (e hylene glycol) dime hyl e he ( e aglyme, Sigma Ald ich, San Louis, MO, USA) in oduced by bubbling a gon in he eac o , and he pa ame e s used we e 100 W, 0.40 mba and 1 h, acco ding o a p e ious wo k [21]. The p ocess was pe o med in pulsed mode wi h on = 20 µs and o = 20 ms. In Table 1, he di e en condi ions o he plasma polyme iza ion can be obse ed. Fi e samples we e used o each ea men (powe peak and ime). Figu e 4. Schema ic ep esen a ion o he me hodology used. The me hodology o plasma ac i a ion p ocess o Ti su aces was ca ied ou as explained by Buxade a e al. [ 10 ]. These samples (n = 30) we e ea ed by adio equency low-p essu e plasma appa a us (Plasma Sys em Fem o, Diene , Ge many) using 13.52 Hz o 10 min ea men , wi h a gon a a p essu e o 0.40 mba (Figu e 5). This ea men was ealized o ac i a e he i anium su ace and, o his eason, was ca ied ou by a gon nonpolyme izing gas. Plasma polyme iza ion was pe o med igh a e plasma ac i a ion in he same eac o wi hou b eaking he acuum. The polyme iza ion p ecu so was e a (e hylene glycol) dime hyl e he ( e aglyme, Sigma Ald ich, San Louis, MO, USA) in oduced by bubbling a gon in he eac o , and he pa ame e s used we e 100 W, 0.40 mba and 1 h, acco ding o a p e ious wo k [ 21 ]. The p ocess was pe o med in pulsed mode wi h on = 20 µ s and o = 20 ms. In Table 1, he di e en condi ions o he plasma polyme iza ion can be obse ed. Fi e samples we e used o each ea men (powe peak and ime). Ma e ials 2022, 15, 7487 5 o 14 Figu e 5. Low-p essu e plasma appa a us used wi h a gon and wi h polyme iza ion ea men . Table 1. Peak powe s and imes used in he PEG polyme iza ion plasma ea men s. Sample Peak Powe (W) Time (min) PEG100-30 100 30 PEG100-60 100 60 PEG150-30 150 30 PEG150-60 150 60 PEG200-30 200 30 PEG200-60 200 60 Roughness was de e mined by means o a whi e ligh in e e ome e mic oscopy (Wyko NT1100, Veeco, New Yo k, NY, USA). Fou samples o each ea men we e analyzed and he measu emen s we e ealized in i e su aces o e alua e he Sa and Pc pa ame e s. Da a analysis was pe o med wi h Wyko Vision 232TM so wa e (Veeco, New Yo k, NY, USA). Based on p e ious es s, he ollowing cu -o alues we e applied: λc = 0.8 mm and λc = 0.25 mm o con ol su aces [29–31]. The alues o he con ac angle (SCA) (Con ac Angle Sys em OCA15plus, Da aphysics, Filde s ad , Ge many) was ca ied ou h ough he sessile d op me hod. The es s we e ealized a 25 °C in an en i onmen al PMMA chambe ha was sa u a ed wi h he s udy liquid o h ee samples o each condi ion. The SCAs we e de e mined wi h ul a-pu e dis illed wa e . A leas h ee measu emen s we e ca ied ou wi h h ee di e en samples in each se ies. The con ac angle measu emen s we e pe o med wi h a con ac angle ideo-based sys em and analyzed wi h SCA20 so wa e [32,33]. X- ay pho oelec on spec oscopy (XPS) was es ed in ul a-high acuum (5.0 × 10−9 mba ) wi h an XR50 Mg anode sou ce ope a ing a 150 W and a Phoibos 150 MCD-9 de ec o (D8 ad ance, SPECS Su ace Nano Analysis GmbH, Be lin, Ge many). C 1 s peak was used as a e e ence. As a e e ence used o compa e he XPS esul s, he heo e ical a omic composi ion o a PEG-amine o molecula weigh 1500 g/mol was calcula ed by coun ing he numbe o a oms p esen in each polyme ic chain [34,35]. These alues we e labeled as heo e ical PEG. Th ee samples o each ea men we e analyzed by XPS. Cy o oxic e ec s o con ol and PEG-coa ed su aces we e analyzed ollowing ISO 10993-5 s anda d on human o eskin ib oblas s (hFFs, Me ck Millipo e Co po a ion, Bed o d, MA, USA) as p e iously epo ed [16,21,36]. Ten samples o each ea men Figu e 5. Low-p essu e plasma appa a us used wi h a gon and wi h polyme iza ion ea men . Ma e ials 2022,15, 7487 5 o 13 Table 1. Peak powe s and imes used in he PEG polyme iza ion plasma ea men s. Sample Peak Powe (W) Time (min) PEG100-30 100 30 PEG100-60 100 60 PEG150-30 150 30 PEG150-60 150 60 PEG200-30 200 30 PEG200-60 200 60 Roughness was de e mined by means o a whi e ligh in e e ome e mic oscopy (Wyko NT1100, Veeco, New Yo k, NY, USA). Fou samples o each ea men we e analyzed and he measu emen s we e ealized in i e su aces o e alua e he Sa and Pc pa ame e s. Da a analysis was pe o med wi h Wyko Vision 232TM so wa e (Veeco, New Yo k, NY, USA). Based on p e ious es s, he ollowing cu -o alues we e applied: λ c = 0.8 mm and λc = 0.25 mm o con ol su aces [29–31]. The alues o he con ac angle (SCA) (Con ac Angle Sys em OCA15plus, Da aphysics, Filde s ad , Ge many) was ca ied ou h ough he sessile d op me hod. The es s we e ealized a 25 ◦ C in an en i onmen al PMMA chambe ha was sa u a ed wi h he s udy liquid o h ee samples o each condi ion. The SCAs we e de e mined wi h ul a-pu e dis illed wa e . A leas h ee measu emen s we e ca ied ou wi h h ee di e en samples in each se ies. The con ac angle measu emen s we e pe o med wi h a con ac angle ideo-based sys em and analyzed wi h SCA20 so wa e [32,33]. X- ay pho oelec on spec oscopy (XPS) was es ed in ul a-high acuum ( 5.0 ×10−9mba ) wi h an XR50 Mg anode sou ce ope a ing a 150 W and a Phoibos 150 MCD-9 de ec o (D8 ad ance, SPECS Su ace Nano Analysis GmbH, Be lin, Ge many). C 1 s peak was used as a e e ence. As a e e ence used o compa e he XPS esul s, he heo e ical a omic composi ion o a PEG-amine o molecula weigh 1500 g/mol was calcula ed by coun ing he numbe o a oms p esen in each polyme ic chain [ 34 , 35 ]. These alues we e labeled as heo e ical PEG. Th ee samples o each ea men we e analyzed by XPS. Cy o oxic e ec s o con ol and PEG-coa ed su aces we e analyzed ollowing ISO 10993-5 s anda d on human o eskin ib oblas s (hFFs, Me ck Millipo e Co po a ion, Bed o d, MA, USA) as p e iously epo ed [ 16 , 21 , 36 ]. Ten samples o each ea men we e s udied. Ex ac s o he samples a concen a ions o 1:1, 1:2, 1:10, 1:100 and 1:1000 we e p epa ed by imme sing he samples in Dulbecco’s modi ied Eagle medium (DMEM, In i ogen, Ca lsbad, CA, USA). A o al o 5000 cells/well on a 96-well issue cul u e polys y ene dish we e in con ac wi h he eluen s o 24 h and hen lysed wi h mam- malian p o ein ex ac ion eagen (mPER, The mo Scien i ic, Wal ham, MA, USA). Cell iabili y was measu ed by he ac i i y o he enzyme lac a e dehyd ogenase (LDH) wi h a Cy o oxici y De ec ion Ki (The mo Scien i ic, USA) as indica ed by he supplie . Bac e ial adhesion es s we e analyzed wi h Spec ococcus Sanguinis CCUG 15915 (Cul u e Collec ion Uni e si y o Gö ebo g (CCUG), Sweden) and Lac obacillus Sali a ius CECT 101 (Colección Española de Cul i os Tipo, Valencia, Spain). Bo h cul u es we e incuba ed om h ee colonies o e nigh a 37 ◦ C be o e he assays in B ain-Hea In usion (BHI, Sha lab SL, Ba celona, Spain) using en samples o each condi ion. A e wa ds, bac e ia suspensions we e dilu ed o an abso bance o 0.20 ± 0.01 a 600 nm using a Laxco Mic oSpek DSM-Cu e e Cell Densi y Me e (Cole Pa me , Ve non Hills, IL, USA), gi ing app oxima ely 1 × 10 8 colony o ming uni s (CFUs)/mL. A o al o 5 µ L o he bac e ial suspension was placed on op o he samples and le o 2 h a 37 ◦ C. A e , samples we e cleaned wice wi h PBS. Adhe en bac e ia we e de ached by o exing he disks o 5 min in 1 mL o PBS. De ached bac e ia we e hen seeded using se ial dilu ions in BHI-aga pla es [ 37 , 38 ]. The pla es we e hen incuba ed o e nigh a 37 ◦ C and he esul ing CFUs we e coun ed. Th ee samples o each condi ion we e s udied. Cell-bac e ia co-cul u e expe imen was an adap a ion om he s udy by Godoy- Galla do e al. [ 39 ]. Fo cell adhesion s udies, 2 × 10 4 cells we e seeded on each sample Ma e ials 2022,15, 7487 6 o 13 and le o 24 h a 37 ◦ C. A e 2 h a 37 ◦ C, samples we e washed h ee imes in o de o elimina e he non-a ached bac e ia, and hFFs in modi ied DMEM (DMEM wi h 2% BHI) a 2×104cells/sample we e seeded and incuba ed o 24 h. The esul s we e s a is ically s udied by S uden ’s - es s, one-way ANOVA ables and Tu key’s mul iple compa ison es s. Using his me hod can e alua e any s a is ically signi ican di e ences be ween he sample g oups. The signi icance di e ences we e when p< 0.05. The s a is ical s udy was ealized by Mini abTM so wa e (Mini ab elease 13.0, Mini ab Inc., S a e College, PA, USA). 3. Resul s Roughness s udies we e ca ied ou on he mini-implan s since, as is well known, oughness is a pa ame e ha will a ec bac e ial coloniza ion. The esul s o he o igi- nal i anium (Ti), i anium ac i a ed by a gon plasma (PA) and hose ea ed wi h PEG polyme iza ion plasma is shown in Table 2. F om hese esul s, no s a is ically signi ican di e ences can be obse ed in any case. The ea men does no a ec he opog aphy o he samples. Table 2. Roughness o he di e en mini-implan s. Sample Ra(µm) Pc(cm−1) Ti 0.33 ±0.10 150.9 ±69 PA 0.35 ±0.20 153.4 ±56 PEG100-30 0.36 ±0.30 152.8 ±59 PEG100-60 0.33 ±0.21 146.9 ±60 PEG150-30 0.43 ±0.12 150.9 ±69 PEG150-60 0.37 ±0.09 150.9 ±69 PEG200-30 0.39 ±0.09 150.9 ±69 PEG200-60 0.32 ±0.12 150.9 ±69 We abili y inc eased wi h he plasma ac i a ion, especially wi h a gon (PA) ea men s. This was ealized by means o a gon a a 100 W peak powe om 64.2 ± 5.5 ◦ o he as- ecei ed i anium o 5.2 ± 1.2 ◦ ea ed wi h a gon, showing an impo an supe -hyd ophilic cha ac e . The con ac angles o he di e en ea men s wi h PEG a e shown in Table 3. Table 3. Con ac angles o he di e en samples wi h PEG. As e isks indica e he s a is ical di e - ence signi icances. Sample Con ac Angle (◦) PEG100-30 12.3 ±0.9 * PEG100-60 13.2 ±1.3 * PEG150-30 18.2 ±1.2 ** PEG150-60 19.1 ±0.8 ** PEG200-30 25.0 ±2.2 *** PEG200-60 25.2 ±2.9 *** S a is ically signi ican di e ences in he con ac angle can be obse ed wi h he di e en peak powe alues applied, bu no di e ences in ea men imes a e obse ed o each peak powe applied. The a omic concen a ion o he elemen s in he ou e su ace was eco ded by XPS and is summa ized in Table 4. Cy ocompa ibili y esul s demons a ed no dec ease a any dilu ion when cul u ed wi h ib oblas s and os eoblas s (Figu es 6and 7, espec i ely). All o he s udied su aces and he plasma polyme iza ion condi ions had cy ocompa ibili y a ios o e 70%. Fo bo h esul s, he e a e no signi ican s a is ical di e ences. The excellen biocompa ibili y o he ea men s can be concluded. Ma e ials 2022,15, 7487 7 o 13 Table 4. A omic concen a ion (in %) o he ca bon, oxygen and i anium amoun p esen on he Ti, and PA. Sample O 1s C 1s Ti 2p Ti 55 ±1 24 ±1 20 ±1 PA 63 ±2 10 ±1 26 ±1 PEG100-30 45 ±2 41 ±1 2 ±1 PEG100-60 54 ±3 37 ±2 5 ±1 PEG150-30 42 ±1 47 ±1 8 ±1 PEG150-60 45 ±1 52 ±1 8 ±1 PEG200-30 48 ±2 44 ±2 7 ±1 PEG200-60 40 ±1 52 ±3 6 ±1 Ma e ials 2022, 15, 7487 8 o 14 Figu e 6. Cell iabili y o he hFFs o he di e en ea men s and di e en dilu ions. Figu e 7. Cell iabili y o he SAOS-2 o he di e en ea men s and di e en dilu ions. Cell adhesion assays wi h hFFs (Figu e 8) showed no di e ence be ween Ti, PA and PEG samples, whe eas, o SAOS-2 (Figu e 9), a sligh ly dec ease was measu ed when he samples we e polyme ized. Figu e 6. Cell iabili y o he hFFs o he di e en ea men s and di e en dilu ions. Ma e ials 2022, 15, 7487 8 o 14 Figu e 6. Cell iabili y o he hFFs o he di e en ea men s and di e en dilu ions. Figu e 7. Cell iabili y o he SAOS-2 o he di e en ea men s and di e en dilu ions. Cell adhesion assays wi h hFFs (Figu e 8) showed no di e ence be ween Ti, PA and PEG samples, whe eas, o SAOS-2 (Figu e 9), a sligh ly dec ease was measu ed when he samples we e polyme ized. Figu e 7. Cell iabili y o he SAOS-2 o he di e en ea men s and di e en dilu ions. Ma e ials 2022,15, 7487 8 o 13 Cell adhesion assays wi h hFFs (Figu e 8) showed no di e ence be ween Ti, PA and PEG samples, whe eas, o SAOS-2 (Figu e 9), a sligh ly dec ease was measu ed when he samples we e polyme ized. Ma e ials 2022, 15, 7487 9 o 14 Figu e 8. Cell adhesion o hFFs on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p < 0.05). Figu e 9. Cell adhesion o SaOS-2 on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p < 0.05). Bac e ial adhesion es s showed a dec eased bac e ial adhesion o all PEG samples, ei he o he Spec ococcus sanguinis and he Lac obacillus sali a ius (Figu es 10 and 11, espec i ely). Ti samples and plasma-ac i a ed samples (PA) we e used as con ols. An inc eased bac e ial adhesion was obse ed o he PA sample compa ed o Ti. Figu e 8. Cell adhesion o hFFs on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p< 0.05). Ma e ials 2022, 15, 7487 9 o 14 Figu e 8. Cell adhesion o hFFs on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p < 0.05). Figu e 9. Cell adhesion o SaOS-2 on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p < 0.05). Bac e ial adhesion es s showed a dec eased bac e ial adhesion o all PEG samples, ei he o he Spec ococcus sanguinis and he Lac obacillus sali a ius (Figu es 10 and 11, espec i ely). Ti samples and plasma-ac i a ed samples (PA) we e used as con ols. An inc eased bac e ial adhesion was obse ed o he PA sample compa ed o Ti. Figu e 9. Cell adhesion o SaOS-2 on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p< 0.05). Bac e ial adhesion es s showed a dec eased bac e ial adhesion o all PEG samples, ei he o he Spec ococcus sanguinis and he Lac obacillus sali a ius (Figu es 10 and 11, espec i ely). Ti samples and plasma-ac i a ed samples (PA) we e used as con ols. An inc eased bac e ial adhesion was obse ed o he PA sample compa ed o Ti. Ma e ials 2022,15, 7487 9 o 13 Ma e ials 2022, 15, 7487 10 o 14 Figu e 10. Bac e ial adhesion on Ti, PA and PEG samples o Spec ococcus sanguinis. As e isks indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05). Figu e 11. Bac e ial adhesion on Ti, PA and PEG samples o Lac obacillus Sali a ius. As e isks indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05). 4. Discussion In his s udy, we aimed o es i polye hylene glycol (PEG) coa ing would signi ican ly p e en he o ma ion o bac e ial bio ilm on he su ace o i anium mini- implan s. The oughness esul s show ha he oughness is no a ec ed by he plasma ea men s. This ac is impo an since i is well known ha oughness a ec s Figu e 10. Bac e ial adhesion on Ti, PA and PEG samples o Spec ococcus sanguinis. As e isks indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p> 0.05). Ma e ials 2022, 15, 7487 10 o 14 Figu e 10. Bac e ial adhesion on Ti, PA and PEG samples o Spec ococcus sanguinis. As e isks indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05). Figu e 11. Bac e ial adhesion on Ti, PA and PEG samples o Lac obacillus Sali a ius. As e isks indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05). 4. Discussion In his s udy, we aimed o es i polye hylene glycol (PEG) coa ing would signi ican ly p e en he o ma ion o bac e ial bio ilm on he su ace o i anium mini- implan s. The oughness esul s show ha he oughness is no a ec ed by he plasma ea men s. This ac is impo an since i is well known ha oughness a ec s Figu e 11. Bac e ial adhesion on Ti, PA and PEG samples o Lac obacillus Sali a ius. As e isks indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p> 0.05). 4. Discussion In his s udy, we aimed o es i polye hylene glycol (PEG) coa ing would signi ican ly p e en he o ma ion o bac e ial bio ilm on he su ace o i anium mini-implan s. The oughness esul s show ha he oughness is no a ec ed by he plasma ea men s. This ac is impo an since i is well known ha oughness a ec s osseoin eg a ion le els, as well as bac e ial p oli e a ion and adhesion [ 40 – 44 ]. These esul s make i possible o op imize he ea men s o ob ain he op imum oughness, knowing ha subsequen plasma ea men s will no a y his impo an cha ac e is ic. The we abili y esul s indica e a e y signi ican a ia ion in he hyd ophilic cha ac e when we ac i a e he su ace wi h a gon. The a ia ion in he con ac angle om 64 ◦ o 5 ◦ makes he su ace supe -hyd ophilic. Rega ding he su ace ea men s wi h PEG, i