Decontaminative Properties of Cold Atmospheric Plasma Treatment on Collagen Membranes Used for Guided Bone Regeneration

Ci a ion: Gülses, A.; Doh mann, L.; Ak as, O.C.; Wagne , J.; Vezi oglu, S.; Tja d s, T.; Ha ig, T.; Lied ke, K.R.; Wil ang, J.; Acil, Y.; e al. Decon amina i e P ope ies o Cold A mosphe ic Plasma T ea men on Collagen Memb anes Used o Guided Bone Regene a ion. J. Func . Bioma e . 2023,14, 372. h ps:// doi.o g/10.3390/j b14070372 Recei ed: 9 June 2023 Re ised: 5 July 2023 Accep ed: 11 July 2023 Published: 14 July 2023 Copy igh : © 2023 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/). Jou nal o Func ional Bioma e ials Communica ion Decon amina i e P ope ies o Cold A mosphe ic Plasma T ea men on Collagen Memb anes Used o Guided Bone Regene a ion Aydin Gülses 1,* , Lina Doh mann 1, O al Cenk Ak as 2, Juliane Wagne 1, Salih Vezi oglu 2,3 , Tim Tja d s 2, To ge Ha ig 2, Kim Rou en Lied ke 4, Jö g Wil ang 1, Yahya Acil 1and Ch is ian Flö ke 1 1Depa men o O al and Maxillo acial Su ge y, Uni e si y Hospi al Schleswig-Hols ein, 24105 Kiel, Ge many; [email p o ec ed] (L.D.); [email p o ec ed] (J.W.); joe [email p o ec ed] (J.W.); [email p o ec ed] (Y.A.); [email p o ec ed] (C.F.) 2Chai o Mul icomponen Ma e ials, Ins i u e o Ma e ials Science, Facul y o Enginee ing, Ch is ian-Alb ech s-Uni e si y o Kiel, 24118 Kiel, Ge many; [email p o ec ed] (O.C.A.); [email p o ec ed] (S.V.); [email p o ec ed] (T.T.); [email p o ec ed] (T.H.) 3Kiel Nano, Su ace and In e ace Science KiNSIS, Kiel Uni e si y, Ch is ian Alb ech s-Pla z 4, 24118 Kiel, Ge many 4Depa men o O hopedics, Uni e si y Hospi al Schleswig-Hols ein, 24105 Kiel, Ge many; [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +49-431-500-26169; Fax: +49-0431-500-26164 Abs ac : Backg ound cold a mosphe ic plasma (CAP) is known o be a su ace- iendly ye an imi- c obial and ac i a ing p ocess o su aces such as i anium. The aim o he p esen s udy was o desc ibe he decon amina ing e ec s o CAP on con amina ed collagen memb anes and hei in lu- ence on he p ope ies o his bioma e ial in i o . Ma e ial and Me hods: A o al o n= 18 Bio-Gide ® (Geis lich Bioma e ials, Baden-Baden, Ge many) memb anes we e examined. The in e en ion g oup was di ided as ollows: n= 6 memb anes we e ea ed o one minu e, and n= 6 memb anes we e ea ed o i e minu es wi h CAP using kINPen ® MED (neoplas ools GmbH, G ei swald, Ge many) wi h an ou pu o 5 W, espec i ely. A non-CAP- ea ed g oup (n= 6) se ed as he con ol. The opog aphic al e a ions we e e alua ed ia X- ay pho oelec on spec oscopy (XPS) and scanning elec on mic oscopy (SEM). A e wa d, he samples we e con amina ed wi h E. aecalis o 6 days, and colony- o ming uni (CFU) coun s and addi ional SEM analyses we e pe o med. The CFUs inc eased wi h CAP ea men ime in ou analyses, bu SEM showed ha he su ace o he memb anes was essen ially ee om bac e ia. Howe e , he deepe laye s showed emaining mic obial conglome a es. Fu he mo e, we showed, ia XPS analysis, ha inc easing he CAP ime signi ican ly enhances he ca bon (ca bonyl g oup) concen a ion, which also co ela es nega i ely wi h he decon amina ing e ec s o CAP. Conclusions: Reac i e ca bonyl g oups o e a po en ial mechanism o inhibi ing he g ow h o E. aecalis on collagen memb anes a e cold a mosphe ic plasma ea men . Keywo ds: ca bon; cold a mosphe ic plasma; E. aecalis; collagen; guided bone egene a ion 1. In oduc ion Collagen is a common bioma e ial in egene a i e medicine due o i s g ea biocompa i- bili y, low an igenici y, p ominen cell a ini y, and biodeg adabili y ha has he po en ial o epai issues and es o e hei physiological unc ion [ 1 – 3 ]. Decellula ized biodeg adable collagen memb anes a e equen ly u ilized in den is y o gene a e a ious compa men s o he epai o c i ical size de ec s. Collagen memb anes a e used in bo h guided bone egene a ion and guided issue egene a ion o ac as an occlusi e ba ie o s op gingi al so issue om g owing in o a pe iodon al o bone de ec . This enables issue egene a ion h ough he un es ic ed p oli e a ion and di e en ia ion o si e-speci ic p ogeni o cells. J. Func . Bioma e . 2023,14, 372. h ps://doi.o g/10.3390/j b14070372 h ps://www.mdpi.com/jou nal/j b J. Func . Bioma e . 2023,14, 372 2 o 10 A bilaye memb ane called Bio-Gide ® is in ended o p o ec a bone de ec om so issue in asion and has been used in den al medicine o se e al decades [ 4 ] in he managemen o pe iodon al and pe i-implan diseases o o enhance he ossi ica ion o bone de ec s o any o igin. In o de o allow o bone ing ow h, Bio-Gide ® ’s ough side mus ace he bone de ec , whe eas he smoo h laye should bea he so issues. The wid h, spacing, and o ien a ion o memb ane ibe s o ib ils, as well as he s i ness o he memb ane, ha e all been ound o in luence cellula beha io s like di e - en ia ion, mig a ion, and p oli e a ion [ 5 – 9 ]. Howe e , memb ane exposu e, which leads o bac e ial con amina ion and o en esul s in he disin eg a ion o he g a , has been epo ed o be he mos common complica ion seconda y o guided bone/ issue egene a- ion [ 10 ], which mos ly necessi a es he comple e emo al o he bioma e ials placed [ 11 ]. Addi ionally, i has been epo ed ha memb ane exposu e a e GBR p ocedu es has a signi ican de imen al in luence on he ou come o bone augmen a ion. Ga cia e al. [ 12 ] ha e epo ed ha he si es wi hou memb ane exposu e achie ed 74% mo e ho izon al bone gain han he si es wi h exposu e. The unc ionaliza ion o collagen-based bioma e ials aims o modi y hei chemical and biological cha ac e is ics and helps o de elop clinical s a egies agains bac e ial con amina ion wi hou impai ing he egene a i e p ope ies. Ta aballi e al. [ 13 ] en iched amine and ca boxylic unc ional g oups on collagen ilms by using N 2 /H 2 and CO 2 plasma and in es iga ed he physico-chemical ea u es o he modi ied su aces as well as hei in i o biocompa ibili y in he p esence o human os eoblas -like cells. Egge s e al. [ 14 ] also showed ha plasma ea men signi ican ly imp o ed os eoblas -like cell iabili y when compa ed o un ea ed cells and p oclaimed ha plasma ea men has a posi i e impac on wound closu e in an in i o se ing. In addi ion o i s egene a i e e ec s, nume ous in es iga ions [ 14 – 18 ] conduc ed o e he pas 20 yea s ha e also es ablished he an ibac e ial ac i i y o plasma ea men . I has been p oclaimed ha cold a mosphe ic plasma (CAP) has he capaci y o en e po ous su aces, e icien ly disin ec ing he in ica e h ee-dimensional su ace [ 19 ]. I was also p o en ha CAP migh inac i a e bio ilm on mic o-s uc u ed i anium su aces wi hou al e ing i s opog aphy. These s udies also demons a ed ha plasma could educe he numbe o li ing bac e ia on he mic o-s uc u ed su ace o den al i anium implan s. Howe e , he e ec s o CAP on di e en bioma e ials emain unclea . Addi ionally, he du a ion o ea men is ano he poin ha has been discussed con o e sially in he li e a u e [20,21]. I is well known ha ee adicals a e a sou ce o oxida i e s ess and a e hos ile o bac e ia [ 20 , 22 ]. Many s udies ha e demons a ed ha an inc eased in acellula le el, which is caused by a di ec CAP je , can ei he eac p ima ily wi h he cell en elope o damage in acellula componen s and subsequen ly lead o bac e ial dea h [ 23 ]. Mos o hese s udies ha e exposed he bac e ial suspension di ec ly unde he CAPs, wi h he bac e ial g ow h being in luenced by he o h igh oxida i e s ess caused by ee adicals [ 23 ]. Howe e , Yang e al. [ 21 ] ecen ly showed ha he ee adicals le on he zi conia su ace can impose oxida i e s ess on bac e ia a e CAP ea men . The e o e, i is impo an o in es iga e he mechanisms o bac e ial dea h on he CAP- ea ed ma e ials o encou age o a oid he use o CAP as a p omising su ace modi ica ion me hod in clinical implan den is y. The e ec s o CAP on collagen memb anes ha e no been e alua ed un il now. The p esen s udy’s objec i e was o desc ibe he decon amina i e e ec s o CAP on con amina ed collagen memb anes and i s in luence on su ace cha ac e is ics in i o. 2. Ma e ials and Me hods 2.1. Memb anes Bio-Gide ® (Geis lich Bioma e ials, Baden-Baden, Ge many), which ac s as a bilaye ba ie made o po cine de mis ype I and III collagen, was selec ed. The memb anes we e cu in 10 ×10 mm samples. A o al o 18 samples we e used. J. Func . Bioma e . 2023,14, 372 3 o 10 2.2. CAP T ea men The ough su ace o each memb ane in he i s g oup (n= 6) was ea ed wi h cold plasma o 1 min. In he second g oup (n= 6), each memb ane was ea ed o 5 min by also using kINPen ® MED (neoplas ools GmbH, G ei swald, Ge many) wi h an ou pu o 5 W, espec i ely (Figu e 1). A non-CAP- ea ed g oup (n= 6) se ed as he con ol. J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 3 o 10 2. Ma e ials and Me hods 2.1. Memb anes Bio-Gide® (Geis lich Bioma e ials, Baden-Baden, Ge many), which ac s as a bilaye ba ie made o po cine de mis ype I and III collagen, was selec ed. The memb anes we e cu in 10×10 mm samples. A o al o 18 samples we e used. 2.2. CAP T ea men The ough su ace o each memb ane in he fi s g oup (n = 6) was ea ed wi h cold plasma o 1 min. In he second g oup (n = 6), each memb ane was ea ed o 5 min by also using kINPen® MED (neoplas ools GmbH, G ei swald, Ge many) wi h an ou pu o 5 W, espec i ely (Figu e 1). A non-CAP- ea ed g oup (n = 6) se ed as he con ol. Figu e 1. T ea men o collagen memb ane wi h cold a mosphe ic plasma using kINPen® MED. 2.3. Su ace Cha ac e iza ion Fo su ace cha ac e iza ion, non-con amina ed memb anes we e CAP ea ed o 1 min and 5 min, espec i ely. A g oup con aining non-CAP- ea ed memb anes se ed as he con ol sample. To emula e he possible changes ega ding cul i a ion, as desc ibed below, he memb anes we e cul i a ed in 10 mL o s e ile nu ien solu ion, which was exchanged e e y 24 h o 6 days. The su ace mo phology o he memb anes was e ealed by scanning elec on mic oscopy (SEM; Sup a55VP-Ca l Zeiss, Obe kochen, Ge many). Fu he mo e, X- ay pho oelec on spec oscopy (XPS; Al anode, 240 W Omic on Nano- Technology GmbH, Taunuss ein, Ge many) measu emen s we e pe o med o de e mine he chemical s a es on he memb ane su ace. All he binding ene gies we e e e enced o he C 1s peak a 285.0  eV o he ad en i ious ca bon on he su ace. 2.4. Bac e ial Con amina ion Bac e ial con amina ion was pe o med as p e iously desc ibed by Flö ke e al. [24]. B iefly, immedia ely a e he CAP ea men , cul i a ion wi h 10 mL o s e ile nu ien solu ion (BHI, B ain–Hea -In usion B o h, Ca l Ro h GmbH + Co. KG, Ka ls uhe, Ge - many) and 100 µL o bac e ial cul u e wi h E. aecalis (ATCC 29,212) was conduc ed a 37 °C o 24 h (He aeus B6060, He aeus Holding GmbH, Hanau, Ge many). A e wa d, he boxes (Eppendo pipe e ip eusable boxes Eppendo AG, Hambu g, Ge many) con ain- ing he models we e s e ilized a 121 °C (Au ocla e Melag Vacukla 24, MELAG Mediz- in echnik oHG, Be lin, Ge many). On he fi s day, he memb anes we e in ec ed wi h 200 mL o s e ile BHI and 100 µL o he o e nigh cul u e and hen incuba ed a 37 °C (Scien- ific C24 Incuba o Shake , New B unswick Scien ific, Edison, NJ, USA). A e 4 h, he Figu e 1. T ea men o collagen memb ane wi h cold a mosphe ic plasma using kINPen®MED. 2.3. Su ace Cha ac e iza ion Fo su ace cha ac e iza ion, non-con amina ed memb anes we e CAP ea ed o 1 min and 5 min, espec i ely. A g oup con aining non-CAP- ea ed memb anes se ed as he con ol sample. To emula e he possible changes ega ding cul i a ion, as desc ibed below, he memb anes we e cul i a ed in 10 mL o s e ile nu ien solu ion, which was exchanged e e y 24 h o 6 days. The su ace mo phology o he memb anes was e ealed by scanning elec on mic oscopy (SEM; Sup a55VP-Ca l Zeiss, Obe kochen, Ge many). Fu he mo e, X- ay pho oelec on spec oscopy (XPS; Al anode, 240 W Omic on Nano- Technology GmbH, Taunuss ein, Ge many) measu emen s we e pe o med o de e mine he chemical s a es on he memb ane su ace. All he binding ene gies we e e e enced o he C 1s peak a 285.0 eV o he ad en i ious ca bon on he su ace. 2.4. Bac e ial Con amina ion Bac e ial con amina ion was pe o med as p e iously desc ibed by Flö ke e al. [ 24 ]. B ie ly, immedia ely a e he CAP ea men , cul i a ion wi h 10 mL o s e ile nu ien so- lu ion (BHI, B ain–Hea -In usion B o h, Ca l Ro h GmbH + Co. KG, Ka ls uhe, Ge many) and 100 µ L o bac e ial cul u e wi h E. aecalis (ATCC 29,212) was conduc ed a 37 ◦ C o 24 h (He aeus B6060, He aeus Holding GmbH, Hanau, Ge many). A e wa d, he boxes (Eppendo pipe e ip eusable boxes Eppendo AG, Hambu g, Ge many) con aining he models we e s e ilized a 121 ◦ C (Au ocla e Melag Vacukla 24, MELAG Medizin echnik oHG, Be lin, Ge many). On he i s day, he memb anes we e in ec ed wi h 200 mL o s e ile BHI and 100 µ L o he o e nigh cul u e and hen incuba ed a 37 ◦ C (Scien i ic C24 Incuba o Shake , New B unswick Scien i ic, Edison, NJ, USA). A e 4 h, he op ical densi y was con olled ia a spec opho ome e (BioPho ome e 6131, Eppendo AG, Hambu g, Ge many) a 600 nm (OD600), which was se o 0.8. The nu ien solu ion was exchanged e e y 24 h wi h 200 mL o s e ile BHI o 6 days. J. Func . Bioma e . 2023,14, 372 4 o 10 2.5. E alua ion o he Bac e ial Decon amina ion The bac e ial decon amina ion o he samples was quan i ied by coun ing he colony- o ming uni s (CFUs) and quali ied by using a scanning elec on mic oscope, as p e iously desc ibed by Flö ke e al. [24]. 2.5.1. Colony-Fo ming Uni s Each memb ane was placed in an Eppendo ube con aining 1 mL o s e ile NaCl solu ion. To de-a ach he bac e ia om he su ace, he memb anes we e placed in an ul asonic ba h (ul asonic ba h B anson 2210R-MT Ul asonic Cleane , B anson Ul asonics Co po a ion, Danbu y/CT, USA) o 20 min. The bac e ial suspension was hen dilu ed o 10–2 and la e o 10–4. A e wa d, he di e en dilu ion le els we e applied on Caso aga pla es. These Caso aga pla es (CASO-Aga Ph.Eu ., Ca l Ro h GmbH + Co. KG, Ka ls uhe, Ge many) we e placed in an incuba o a 37 ◦ C (He aeus B6060 incuba o , He aeus Holding GmbH, Hanau, Ge many). A e 24 h, he colony- o ming uni s we e coun ed wi h a ge m coun e (Ge m coun e BZG 25 om WTW, Xylem Analy ics Ge many Sales GmbH & Co. KG, Weilheim, Ge many). 2.5.2. Scanning Elec on Mic oscope The memb anes we e washed o 1 min wi h PBS (phospha e-bu e ed saline solu ion, Dulbeco, Bioch om GmbH, Be lin, Ge many) and la e wi h 1 mL o 4% glu a aldehyde. A e wa d, he memb anes we e washed h ee imes o 5 min, each ime wi h PBS. The dehyd a ion was ca ied ou by means o an ascending se ies o 30%, 50%, 70%, 90%, and 100% e hanol. The memb anes we e hen ai -d ied un il he e hanol was comple ely e apo a ed. The memb anes we e hen a ached o SEM sample pla es (Aga Scien i ic L d., S ans ed, Essex, UK) and s o ed o e nigh in a desicca o (E ich Eydam KG, Kiel, Ge many). Gold spu e ing a a hickness o 5 nm (BAL-TEC SCD 500, Leica Mic osys ems GmbH, We zla , Ge many) and examina ion using a scanning elec on mic oscope (Philips XL 30 ESEM, Philips GmbH Ma ke DACH, Hambu g, Ge many) was pe o med. Fo each memb ane, i e co esponding a eas a a magni ica ion o 5000 × and i e a eas a a magni ica ion o 8000×we e eco ded. 2.6. S a is ical Analysis Desc ip i e s a is ical analysis was ca ied ou using IBM, SPSS, S a is ics e sion 24.0 o Windows (IBM GmbH, Ehningen, Ge many). A pai ed - es was conduc ed o e alua e he s a is ical di e ences be ween he g oups (non- ea ed s. CAP o 1 min. and non- ea ed s. CAP o 5 min). The signi icance le el was se o (p< 0.05). 3. Resul s 3.1. SEM Analysis o he Su aces The SEM images show ha he su ace mo phology changed undamen ally a e ea men wi h CAP. Figu e 2a shows he smoo h su ace mo phology o he un ea ed collagen memb ane. I can be clea ly seen om Figu e 2b ha he memb ane su ace s ill had many collagen ib ils ha we e closely in e wined a e CAP ea men o 1 min. These ib il bundles e eal a ypical pe iodic banding pa e n. Howe e , ollowing CAP ea men o 5 min, he caudal side clea ly shows ci cula i egula i ies and discon inui ies (Figu e 2c). He e, CAP migh chemically elimina e/deg ade he ma e ials in hose egions. 3.2. XPS Analysis In o de o iden i y he elemen al composi ion and chemical s a e o he memb ane su ace, XPS spec a o he samples (be o e and a e CAP ea men ) we e eco ded, as shown in Figu e 3. As expec ed o he collagen memb ane, he wide-scan XPS spec um showed he p esence o ca bon (C), ni ogen (N), and oxygen (O) on he sample su ace wi hou any ex e nal con amina ion (Figu e 3a). Addi ionally, he XPS su ey shows ha a e CAP ea men , no majo con amina ions om he p epa a ion and p ocessing s eps J. Func . Bioma e . 2023,14, 372 5 o 10 we e obse ed (Figu e 3a). Howe e , inc easing CAP ime signi ican ly enhanced he ca bon concen a ion om 27.68% o 40.39% (ca bonyl g oup: C=O; co esponding peak posi ion a ound 288.5 eV) on he su ace (Figu e 3b). This migh be explained by he ac ha CAP can s a deg ading he o ganic ma e ials in he ib ils, which causes a signi ican inc ease in he ca bon con en on he su ace. Besides, he inco po a ion o A gon due o he plasma pen is negligible and was no obse ed. J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 5 o 10 Figu e 2. SEM image o (a) un ea ed collagen memb ane and ollowing CAP ea men o (b) 1 min and (c) 5 min. 3.2. XPS Analysis In o de o iden i y he elemen al composi ion and chemical s a e o he memb ane su ace, XPS spec a o he samples (be o e and a e CAP ea men ) we e eco ded, as shown in Figu e 3. As expec ed o he collagen memb ane, he wide-scan XPS spec um showed he p esence o ca bon (C), ni ogen (N), and oxygen (O) on he sample su ace wi hou any ex e nal con amina ion (Figu e 3a). Addi ionally, he XPS su ey shows ha a e CAP ea men , no majo con amina ions om he p epa a ion and p ocessing s eps we e obse ed (Figu e 3a). Howe e , inc easing CAP ime significan ly enhanced he ca - bon concen a ion om 27.68% o 40.39% (ca bonyl g oup: C=O; co esponding peak po- si ion a ound 288.5 eV) on he su ace (Figu e 3b). This migh be explained by he ac ha CAP can s a deg ading he o ganic ma e ials in he fib ils, which causes a significan inc ease in he ca bon con en on he su ace. Besides, he inco po a ion o A gon due o he plasma pen is negligible and was no obse ed. Figu e 3. (a) Wide scan and (b) high- esolu ion ca bon XPS spec a o un ea ed and ea ed samples o diffe en ime in e als. The ela i e dis ibu ion o ca bon unc ional g oups om in eg a ion and he ela- i e and absolu e in ensi ies o he ca bon unc ional g oups ep esen ing he fi ing unc- ions a e shown in Table 1. The peak in ensi y was de e mined as he a ea be ween he peak and he baseline. Figu e 2. SEM image o ( a ) un ea ed collagen memb ane and ollowing CAP ea men o ( b ) 1 min and (c) 5 min. J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 5 o 10 Figu e 2. SEM image o (a) un ea ed collagen memb ane and ollowing CAP ea men o (b) 1 min and (c) 5 min. 3.2. XPS Analysis In o de o iden i y he elemen al composi ion and chemical s a e o he memb ane su ace, XPS spec a o he samples (be o e and a e CAP ea men ) we e eco ded, as shown in Figu e 3. As expec ed o he collagen memb ane, he wide-scan XPS spec um showed he p esence o ca bon (C), ni ogen (N), and oxygen (O) on he sample su ace wi hou any ex e nal con amina ion (Figu e 3a). Addi ionally, he XPS su ey shows ha a e CAP ea men , no majo con amina ions om he p epa a ion and p ocessing s eps we e obse ed (Figu e 3a). Howe e , inc easing CAP ime significan ly enhanced he ca - bon concen a ion om 27.68% o 40.39% (ca bonyl g oup: C=O; co esponding peak po- si ion a ound 288.5 eV) on he su ace (Figu e 3b). This migh be explained by he ac ha CAP can s a deg ading he o ganic ma e ials in he fib ils, which causes a significan inc ease in he ca bon con en on he su ace. Besides, he inco po a ion o A gon due o he plasma pen is negligible and was no obse ed. Figu e 3. (a) Wide scan and (b) high- esolu ion ca bon XPS spec a o un ea ed and ea ed samples o diffe en ime in e als. The ela i e dis ibu ion o ca bon unc ional g oups om in eg a ion and he ela- i e and absolu e in ensi ies o he ca bon unc ional g oups ep esen ing he fi ing unc- ions a e shown in Table 1. The peak in ensi y was de e mined as he a ea be ween he peak and he baseline. Figu e 3. ( a ) Wide scan and ( b ) high- esolu ion ca bon XPS spec a o un ea ed and ea ed samples o di e en ime in e als. The ela i e dis ibu ion o ca bon unc ional g oups om in eg a ion and he ela i e and absolu e in ensi ies o he ca bon unc ional g oups ep esen ing he i ing unc ions a e shown in Table 1. The peak in ensi y was de e mined as he a ea be ween he peak and he baseline. Table 1. Rela i e dis ibu ion o ca bon unc ional g oups om in eg a ion. C–H, C–C/a % C–O, C–N/a % C=O/a % CaC2o Cha ging Un ea ed 15.18 57.14 27.68 NaN 5 min 31.67 28.12 37.67 2.53 1 min 23.02 32.71 40.39 3.88 J. Func . Bioma e . 2023,14, 372 6 o 10 3.3. Colony-Fo ming Uni s The esul s o he pai ed- es indica ed ha he e is a di e ence be ween he non- ea ed (8.7 ± 11.2) and CAP- ea ed (21.3 ± 8.6) memb anes ( ea men o 1 min) ega ding he CFU alues (p= 0.133), bu e en i his medium di e ence is p esen , i ell sho o he signi icance le el. A e CAP ea men o 5 min, a signi ican di e ence be ween he o ma ion o he CFUs (24.0 ±4.4) could be de ec ed (p= 0.010, Figu e 4). J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 6 o 10 Table 1. Rela i e dis ibu ion o ca bon unc ional g oups om in eg a ion. C–H, C–C/a % C–O, C–N/a % C=O/a % CaC 2 o Cha ging Un ea ed 15.18 57.14 27.68 NaN 5 min 31.67 28.12 37.67 2.53 1 min 23.02 32.71 40.39 3.88 3.3. Colony-Fo ming Uni s The esul s o he pai ed- es indica ed ha he e is a diffe ence be ween he non- ea ed (8.7 ± 11.2) and CAP- ea ed (21.3 ± 8.6) memb anes ( ea men o 1 min) ega d- ing he CFU alues (p = 0.133), bu e en i his medium diffe ence is p esen , i ell sho o he significance le el. A e CAP ea men o 5 min, a significan diffe ence be ween he o ma ion o he CFUs (24.0 ± 4.4) could be de ec ed (p = 0.010, Figu e 4). Figu e 4. The esul s o he o ma ion o colony- o ming uni s o he un ea ed memb anes (Con- ol; 8.7 ± 11.2), he memb anes ha we e ea ed wi h CAP o 1 min (21.3 ± 8.6), and he memb anes ha we e ea ed wi h CAP o 5 min (24.0 ± 4.4). A pai ed - es was pe o med o es o signifi- cance. A significan diffe ence was de ec ed be ween un ea ed and CAP- ea ed memb anes when ea men was pe o med o 5 min (p = 0.010). * showing he s a is ically significance. 3.4. SEM Analysis Six days a e con amina ion, many mic o-o ganisms and conglome a es on he su - aces could be seen (Figu e 5a,b). A e CAP ea men o 1 (Figu e 6a,b) and 5 (Figu e 7a–c) minu es, he su ace o he memb anes was ee om bac e ia; howe e , he deepe laye s showed he emaining conglome a es. Figu e 5. Six days a e con amina ion, he su ace (a) was ully co e ed wi h mic o-o ganisms and conglome a es (b). Figu e 4. The esul s o he o ma ion o colony- o ming uni s o he un ea ed memb anes (Con ol; 8.7 ± 11.2), he memb anes ha we e ea ed wi h CAP o 1 min (21.3 ± 8.6), and he memb anes ha we e ea ed wi h CAP o 5 min (24.0 ± 4.4). A pai ed - es was pe o med o es o signi icance. A signi ican di e ence was de ec ed be ween un ea ed and CAP- ea ed memb anes when ea men was pe o med o 5 min (p= 0.010). * showing he s a is ically signi icance. 3.4. SEM Analysis Six days a e con amina ion, many mic o-o ganisms and conglome a es on he su - aces could be seen (Figu e 5a,b). A e CAP ea men o 1 (Figu e 6a,b) and 5 (Figu e 7a–c) minu es, he su ace o he memb anes was ee om bac e ia; howe e , he deepe laye s showed he emaining conglome a es. J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 6 o 10 Table 1. Rela i e dis ibu ion o ca bon unc ional g oups om in eg a ion. C–H, C–C/a % C–O, C–N/a % C=O/a % CaC 2 o Cha ging Un ea ed 15.18 57.14 27.68 NaN 5 min 31.67 28.12 37.67 2.53 1 min 23.02 32.71 40.39 3.88 3.3. Colony-Fo ming Uni s The esul s o he pai ed- es indica ed ha he e is a diffe ence be ween he non- ea ed (8.7 ± 11.2) and CAP- ea ed (21.3 ± 8.6) memb anes ( ea men o 1 min) ega d- ing he CFU alues (p = 0.133), bu e en i his medium diffe ence is p esen , i ell sho o he significance le el. A e CAP ea men o 5 min, a significan diffe ence be ween he o ma ion o he CFUs (24.0 ± 4.4) could be de ec ed (p = 0.010, Figu e 4). Figu e 4. The esul s o he o ma ion o colony- o ming uni s o he un ea ed memb anes (Con- ol; 8.7 ± 11.2), he memb anes ha we e ea ed wi h CAP o 1 min (21.3 ± 8.6), and he memb anes ha we e ea ed wi h CAP o 5 min (24.0 ± 4.4). A pai ed - es was pe o med o es o signifi- cance. A significan diffe ence was de ec ed be ween un ea ed and CAP- ea ed memb anes when ea men was pe o med o 5 min (p = 0.010). * showing he s a is ically significance. 3.4. SEM Analysis Six days a e con amina ion, many mic o-o ganisms and conglome a es on he su - aces could be seen (Figu e 5a,b). A e CAP ea men o 1 (Figu e 6a,b) and 5 (Figu e 7a–c) minu es, he su ace o he memb anes was ee om bac e ia; howe e , he deepe laye s showed he emaining conglome a es. Figu e 5. Six days a e con amina ion, he su ace (a) was ully co e ed wi h mic o-o ganisms and conglome a es (b). Figu e 5. Six days a e con amina ion, he su ace ( a ) was ully co e ed wi h mic o-o ganisms and conglome a es (b). J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 7 o 10 Figu e 6. CAP ea men o 1 min esul ed in a dec ease in bac e ial coloniza ion on he su ace o he collagen ma e ial. The des uc ion o he bac e ial memb anes could be de ec ed (a). In he deepe laye s, he bac e ial conglome a es we e s ill ema kable (b). Figu e 7. SEM analysis a e CAP ea men o 5 min esul ed in he decon amina ion o he mem- b ane su ace; howe e , bac e ial conglome a es emained in he deepe laye s (a–c). 4. Discussion Wi hin he las wo decades, he plasma ea men o den al bioma e ials has become he main subjec o many s udies. The majo i y o he CAP s a egies a e based on he managemen o he imp o emen o cell adhesion on i an and zi conia su aces and a e aimed a op imizing he osseoin eg a ion p ocess [25]. On he o he hand, CAP ea men has also been shown o be an efficien me hod o he managemen o pe i-implan i is when combined wi h mechanical deb idemen [24], hanks o i s an imic obial effec s [26]. S imula o y effec s and CAP-induced cell mobili y seem o play a majo ole in highe cell iabili y and imp o ed cell mig a ion [27,28]. On he o he hand, i is e y well known ha CAP ea men can imp o e he su ace we abili y o bioma e ials [29]. Duske e al. [30] showed ha CAP ea men educes he con ac angle and suppo s he sp eading o os eoblas ic cells and sugges ed ha he applica ion o cold plasma may be suppo i e in he ea men o pe i-implan lesions and may imp o e he p ocess o e-osseoin eg a ion. Simila ly, Wagne e al. [24] p oclaimed ha he healing capaci y p o ided h ough CAP ea men could enhance he osseoin eg a ion o den al implan s and has he po en ial o se e as an effec i e ea men op ion in pe iimplan i is he apy. Howe e , i is e y well documen ed ha su aces wi h a highe we abili y show highe bac e ial adhesion and/o bac e ial coloniza ion [31]. The e o e, despi e i s an ibac e ial effec s, al e a ions o su ace opog aphy we e co ela ed wi h bac e ial adhesion. The aim o pe i-implan i is he apy is o decon amina e he implan su ace and, i possible, induce he egene a ion o he pe i-implan ed issues. Fo his eason, i migh be specula ed ha he e exis s a dange in p oposing CAP in he managemen o pe i-implan i is due o opog aphical al e a ions a e CAP ea men and he possible influence o his on bac e ial adhesion. Memb ane exposu e, ollowing guided bone/ issue egene a ion, p esen s a g ea challenge o he den al clinician; hus, bac e ial con amina ion can lead o an in ec ion and, he e o e, necessi a es he comple e emo al o any placed bioma e ials. In ecen yea s, no el s a egies o enhance he an imic obial effec ha e been p oposed by changing Figu e 6. CAP ea men o 1 min esul ed in a dec ease in bac e ial coloniza ion on he su ace o he collagen ma e ial. The des uc ion o he bac e ial memb anes could be de ec ed ( a ). In he deepe laye s, he bac e ial conglome a es we e s ill ema kable (b). J. Func . Bioma e . 2023,14, 372 7 o 10 J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 7 o 10 Figu e 6. CAP ea men o 1 min esul ed in a dec ease in bac e ial coloniza ion on he su ace o he collagen ma e ial. The des uc ion o he bac e ial memb anes could be de ec ed (a). In he deepe laye s, he bac e ial conglome a es we e s ill ema kable (b). Figu e 7. SEM analysis a e CAP ea men o 5 min esul ed in he decon amina ion o he mem- b ane su ace; howe e , bac e ial conglome a es emained in he deepe laye s (a–c). 4. Discussion Wi hin he las wo decades, he plasma ea men o den al bioma e ials has become he main subjec o many s udies. The majo i y o he CAP s a egies a e based on he managemen o he imp o emen o cell adhesion on i an and zi conia su aces and a e aimed a op imizing he osseoin eg a ion p ocess [25]. On he o he hand, CAP ea men has also been shown o be an efficien me hod o he managemen o pe i-implan i is when combined wi h mechanical deb idemen [24], hanks o i s an imic obial effec s [26]. S imula o y effec s and CAP-induced cell mobili y seem o play a majo ole in highe cell iabili y and imp o ed cell mig a ion [27,28]. On he o he hand, i is e y well known ha CAP ea men can imp o e he su ace we abili y o bioma e ials [29]. Duske e al. [30] showed ha CAP ea men educes he con ac angle and suppo s he sp eading o os eoblas ic cells and sugges ed ha he applica ion o cold plasma may be suppo i e in he ea men o pe i-implan lesions and may imp o e he p ocess o e-osseoin eg a ion. Simila ly, Wagne e al. [24] p oclaimed ha he healing capaci y p o ided h ough CAP ea men could enhance he osseoin eg a ion o den al implan s and has he po en ial o se e as an effec i e ea men op ion in pe iimplan i is he apy. Howe e , i is e y well documen ed ha su aces wi h a highe we abili y show highe bac e ial adhesion and/o bac e ial coloniza ion [31]. The e o e, despi e i s an ibac e ial effec s, al e a ions o su ace opog aphy we e co ela ed wi h bac e ial adhesion. The aim o pe i-implan i is he apy is o decon amina e he implan su ace and, i possible, induce he egene a ion o he pe i-implan ed issues. Fo his eason, i migh be specula ed ha he e exis s a dange in p oposing CAP in he managemen o pe i-implan i is due o opog aphical al e a ions a e CAP ea men and he possible influence o his on bac e ial adhesion. Memb ane exposu e, ollowing guided bone/ issue egene a ion, p esen s a g ea challenge o he den al clinician; hus, bac e ial con amina ion can lead o an in ec ion and, he e o e, necessi a es he comple e emo al o any placed bioma e ials. In ecen yea s, no el s a egies o enhance he an imic obial effec ha e been p oposed by changing Figu e 7. SEM analysis a e CAP ea men o 5 min esul ed in he decon amina ion o he memb ane su ace; howe e , bac e ial conglome a es emained in he deepe laye s (a–c). 4. Discussion Wi hin he las wo decades, he plasma ea men o den al bioma e ials has become he main subjec o many s udies. The majo i y o he CAP s a egies a e based on he managemen o he imp o emen o cell adhesion on i an and zi conia su aces and a e aimed a op imizing he osseoin eg a ion p ocess [ 25 ]. On he o he hand, CAP ea men has also been shown o be an e icien me hod o he managemen o pe i-implan i is when combined wi h mechanical deb idemen [24], hanks o i s an imic obial e ec s [26]. S imula o y e ec s and CAP-induced cell mobili y seem o play a majo ole in highe cell iabili y and imp o ed cell mig a ion [ 27 , 28 ]. On he o he hand, i is e y well known ha CAP ea men can imp o e he su ace we abili y o bioma e ials [ 29 ]. Duske e al. [ 30 ] showed ha CAP ea men educes he con ac angle and suppo s he sp eading o os- eoblas ic cells and sugges ed ha he applica ion o cold plasma may be suppo i e in he ea men o pe i-implan lesions and may imp o e he p ocess o e-osseoin eg a ion. Simila ly, Wagne e al. [ 24 ] p oclaimed ha he healing capaci y p o ided h ough CAP ea men could enhance he osseoin eg a ion o den al implan s and has he po en ial o se e as an e ec i e ea men op ion in pe iimplan i is he apy. Howe e , i is e y well documen ed ha su aces wi h a highe we abili y show highe bac e ial adhesion and/o bac e ial coloniza ion [ 31 ]. The e o e, despi e i s an ibac e ial e ec s, al e a ions o su ace opog aphy we e co ela ed wi h bac e ial adhesion. The aim o pe i-implan i is he apy is o decon amina e he implan su ace and, i possible, induce he egene a ion o he pe i-implan ed issues. Fo his eason, i migh be specula ed ha he e exis s a dange in p oposing CAP in he managemen o pe i-implan i is due o opog aphical al e a ions a e CAP ea men and he possible in luence o his on bac e ial adhesion. Memb ane exposu e, ollowing guided bone/ issue egene a ion, p esen s a g ea challenge o he den al clinician; hus, bac e ial con amina ion can lead o an in ec ion and, he e o e, necessi a es he comple e emo al o any placed bioma e ials. In ecen yea s, no el s a egies o enhance he an imic obial e ec ha e been p oposed by changing he memb ane su ace o inco po a ing long- e m eleased an imic obials [ 32 ]. The cu en ex i o s udy aimed o answe a clinical ques ion based on he ollowing hypo hesis: can CAP ea men allow o bac e ial decon amina ion in he case o memb ane exposu e a e guided bone/ issue egene a ion? Despi e i s limi a ions, such as he use o an ex- i o expe imen al model wi h mono-bac e ial decon amina ion, he cu en s udy showed ha CAP ea men could educe bac e ial coloniza ion only on he su ace o he collagen memb ane. Despi e success ul decon amina ion o he su ace o he memb ane, quan- i ica ion ia colony- o ming uni coun s showed an inc ease a e CAP ea men , which also co ela es signi ican ly wi h he du a ion o CAP applica ion. This e ec migh be a ibu ed o he al e a ions o he opog aphy and he inc eased ca bonyl con en o he ma e ial a e CAP ea men . F om he pe spec i e o he ma e ials sciences, plasma ea men is o en used o modi y he su ace p ope ies o polyme ilms since i o e s nume ous ad an ages o e con en ional su ace modi ica ion echniques [ 33 ]. Azam e al. s a ed ha he a ailabili y o unc ional g oups (N–H and C–H) migh p omo e adhesion on den al bioma e ials a e plasma ea men [ 34 ]. Simila ly, Mo en e al. showed ha plasma ea men leads o he J. Func . Bioma e . 2023,14, 372 8 o 10 inco po a ion o C–O, C=O, and O–C=O g oups on polyme s [ 35 ]. In a ecen a icle, Yang e al. demons a ed ha eac i e oxygen species o e a po en ial mechanism o inhibi ing he g ow h o S. mu ans on zi conia su aces ea ed wi h cold a mosphe ic plasma. The inc ease in bac e ial p oli e a ion in he CAP- ea ed g oups e i ied by CFU assays in ou s udy could be explained by he ac ha su ace ca bonyl (C=O) g oups end o p omo e bac e ial g ow h and ac i i y. 5. Conclusions Cold a mosphe ic plasma undamen ally changes he mo phology o collagen mem- b anes, and bac e ial coloniza ion could be physically elimina ed om he su ace. How- e e , he deepe laye s o he memb anes, especially, do no unde go decon amina ion a all and seem o build a mic obial ese oi . The e o e, he decon amina ion o collagen memb ane su aces using a plasma pen migh no be an op ion in he managemen o exposed memb anes ha a e used o guided bone/ issue egene a ion. Au ho Con ibu ions: All samples we e p epa ed and ea ed by C.F., A.G., L.D. and J.W. (Juliane Wagne ). Documen a ion was pe o med by K.R.L. and O.C.A., S.V., T.H. and T.T. assessed he su ace cha ac e is ics. The me hodology was e iewed by Y.A. and J.W. (Jö g Wil ang). Quan i ica ion o bac e ial coloniza ion was conduc ed by A.G. and C.F. The manusc ip was p epa ed by J.W. (Juliane Wagne ) and A.G. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: We acknowledge inancial suppo om Land Schleswig-Hols ein wi hin he unding p og ams Open Access Publika ions onds. The wo k o T.H. was suppo ed by he Ge man Resea ch Founda ion o unding h ough he RTG 2154 ia p ojec P4. Da a A ailabili y S a emen : The da ase s used and/o analyzed du ing he cu en s udy a e a ailable om he au ho ([email p o ec ed]) upon easonable eques . Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. Mau e , T.; S o el, M.H.; Belyae , Y.; S ie el, N.G.; Vidondo, B.; Küke , S.; Mogel, H.; Schä e , B.; Balme , J. 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