Effect of gentamicin sulfate and polymeric polyethylene glycol coating on the degradation and cytotoxicity of iron-based biomaterials

E ec o Gen amicin Sul a e and Polyme ic Polye hylene Glycol Coa ing on he Deg ada ion and Cy o oxici y o I on-Based Bioma e ials Ma ina Pe áko á, Radka Go ejo á, Jana Shepa, Ján Macko, Mi iam Kupko á, Ma ej Micusík, Ma ej Baláz, Vanda Hajducko á, Pa ícia Hudeco á, Ma in Kozá , Ba bo a Sisko á, Pe Sáha, and Rená a O inako á* Ci e This: ACS Omega 2024, 9, 27113−27126 Read Online ACCESS Me ics & Mo e A icle Recommenda ions ABSTRACT: The wo k is ocused on he deg ada ion, cy o oxici y, and an ibac e ial p ope ies, o i on-based bioma e ials wi h a bioac i e coa ing laye . The oam and he compac i on samples we e coa ed wi h a polye hylene glycol (PEG) polyme laye wi hou and wi h gen amicin sul a e (PEG + Ge). The co osion p ope ies o coa ed and uncoa ed samples we e s udied using he deg ada ion es ing in Hanks’ solu ion a 37 °C. The elec ochemical and s a ic imme sion co osion es s e ealed ha he PEG- coa ed samples co oded as e han samples wi h he bioac i e PEG + Ge coa ing and uncoa ed samples. The oam samples co oded as e compa ed wi h he compac samples. To de e mine he cy o oxici y, cell iabili y was moni o ed in he p esence o po ous oam and compac i on samples. The an ibac e ial ac i i y o he samples wi h PEG and PEG + Ge agains Esche ichia coli CCM 3954 and S aphylococcus au eus CCM 4223 s ains was also es ed. Tes ed PEG + Ge samples showed signi ican an ibac e ial ac i i y agains bo h bac e ial s ains. The e o e, he biodeg adable i on-based ma e ials wi h a bioac i e coa ing could be a sui able successo o he me al ma e ials s udied hus a as well as he ma e ials used in he ield o medicine. 1. INTRODUCTION The e is a long- e m g owing demand o o hopedic implan s in he wo ld, mainly due o he inc eased numbe o ac u es and inju ies, especially in he elde ly popula ion. These inju ies signi ican ly a ec he quali y o li e o pa ien s; he e o e, bone implan s ha e become a sough -a e g oup o implan s. 1,2 Me als play an impo an ole in he human body. In he o m o implan s, me als a e used, o example, in bone join eplacemen s and den al implan s. Mos me al implan s ind applica ion in o hopedic su ge y due o hei ad an ages, such as highe ensile s eng h and du abili y compa ed o ce amics and polyme s. Me als, such as s ainless s eel, Co−C alloys, o i anium and i s alloys, a e s ill used in biomedical pe manen implan s. 3−5 Biodeg adable ma e ials ha e become a end in ecen yea s. Thei bigges ad an age is con olled eso p ion di ec ly in he pa ien ’s body. Me allic biodeg adable bioma e ials ha e good mechanical p ope ies, bu hey a e made o me als ha can be eleased in a ce ain amoun due o he co osi e en i onmen o body luids. The e o e, in addi ion o he possible oxici y o he ma e ial, he po en ial oxici y o i s deg ada ion p oduc s mus also be conside ed. These p ope - ies a ec he li ing sys em in which hey a e implan ed and can lead o de e io a ion o he implan ’s p ope ies, esul ing in damage o he implan i sel and consequen ly o a educ ion in i s biocompa ibili y. 6−9 I on as a bioma e ial is compa ible wi h human physiology, has a simila densi y o human bone, as well as good mechanical compa ibili y. 10 This wo k ocuses on i on po ous as well as compac ma e ials because, despi e he indispu able ad an ages o po ous ma e ials, some p ope ies o compac i on can be used in load-bea ing applica ions. In he same way, he use o compac ma e ials compa ed o oams is ad an ageous, o example, in some es s as e e ence ma e ials, o example, om he poin o iew o biocompa ibili y es ing. Compac i on exhibi s a Young’s modulus o 210 GPa. Howe e , Young’s modulus is in he ange o 10−20 GPa o Recei ed: Janua y 31, 2024 Re ised: May 17, 2024 Accep ed: May 24, 2024 Published: June 12, 2024 A icle h p://pubs.acs.o g/jou nal/acsod © 2024 The Au ho s. Published by Ame ican Chemical Socie y 27113 h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 This a icle is licensed unde CC-BY-NC-ND 4.0 Downloaded ia TOMAS BATA UNIV IN ZLIN on Oc obe 3, 2024 a 14:46:56 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. cancellous bone and 3 ×10−4−3×10−3GPa o abecula bone. The use o compac i on ma e ials in he o m o implan s can esul in s ess shielding mainly due o he di e en s i ness o he implan and he bone, which is p ecisely wha he p oduc ion and use o po ous i on ma e ials a e ying o p e en . Po ous i on-based ma e ials wi h po osi y in he ange o 45.6−86.9% exhibi a comp essi e modulus o elas ici y in he ange o 218−845 MPa, which is close o he alues o abecula bone. The size and mu ual connec i i y o he po es ep esen he key ac o s ha in luence he biological and mechanical p ope ies o po ous ma e ials. 11,12 The po ous s uc u e o deg adable ma e ials is used mainly due o he simila i y wi h he s uc u e and p ope ies o human bone, as e biological deg ada ion, as well as he abili y o anspo damaged issues o body luids, which can signi ican ly sho en he necessa y egene a ion ime. 10,13 A sui able app oach in he design o biodeg adable implan s o medical pu poses may be o combine he good mechanical p ope ies o me allic bioma e ials wi h he biocompa ibili y and deg ada ion p ope ies o polyme ic bioma e ials by applying polyme ic coa ings o me als. Due o i s high solubili y in aqueous media, good biocompa ibili y, biodeg ad- abili y, hyd ophilici y, and mechanical p ope ies simila o hose o some so issues, polye hylene glycol (PEG) is sui able o biomedical applica ions including su ace mod- i ica ion, bioconjuga ion, d ug deli e y, and issue enginee ing. Mo eo e , PEG can be a ached o he su ace o d ug- encapsula ing ma e ials o inc ease s abili y and solubili y in i o and educe he a e o deg ada ion om he bloods eam, he eby op imizing he e icacy o he adminis e ed d ug. 14−17 Su ace coa ing can also imp o e an ibac e ial p ope ies o p e en any pos ope a i e in ec ions, 18,19 which a e among he mos common complica ions a e su ge y and may be dange ous o pa ien s. Bone in ec ions a e among he main p oblems ha occu when a o eign body is implan ed in he physiological en i onmen . The use o d ug deli e y sys ems speci ically is an e ec i e means o ea ing local in ec- ions. 20−22 An ibio ics such as ancomycin o gen amicin ha e been popula ized o local an ibio ic adminis a ion by inco po a ing hese an ibio ics in o bone cemen used o ix p os he ic implan s. Howe e , se e al ecen s udies ha e epo ed ha hese an ibio ic-loaded bone cemen s a e no e y e ec i e. Con olled an ibio ic- elease coa ings based on biodeg adable ma e ials a e, he e o e, becoming a possible al e na i e. Biodeg adable coa ings wi h an an ibio ic con en on he su ace o he implan s suppo he elease o he d ug du ing he deg ada ion o he su ace laye s ha each he in e ace o he implan su ace and issue. 23 Gen amicin sul a e is an aminoglycoside an ibio ic used mainly o deal wi h bone in ec ions due o i s ela i ely b oad an imic obial spec um and high he mos abili y. 21,24,25 Resea ch by Nichol e al. e ealed ha he addi ion o gen amicin o a monolaye o ganic−ino ganic hyb id sol−gel coa ing comple ely e adica ed plank onic bac e ia as well as bio ilms o a panel o clinically ele an s aphylococci, while such a coa ing did no in e e e wi h bone healing. 26 Likewise, high- and long- e m doses o gen amicin can igge se ious ad e se eac ions in he su ounding ne es, so i is impo an o choose only he necessa y concen a ion o he d ug o he gi en ime. 27 Since bac e ial coloniza ion usually occu s in he i s hou s a e ma e ial implan a ion, sho - e m sys emic p ophylaxis is as e ec i e as long- e m p e en ion. In ac , a sho - e m local d ug deli e y sys em can mee he equi e- men s o p e en local in ec ion while limi ing possible long- e m ad e se side e ec s. 28 The applica ion o polyme ic and bioac i e an ibio ic coa ings (con aining gen amicin sul a e) on i on subs a es ep esen s a new concep o imp o ing deg ada ion and biocompa ibili y. The combina ion o an i on-based sample, a polyme PEG coa ing, and an an ibio ic (gen amicin sul a e) also ep esen s a p omising concep in e ms o an ibac e ial p ope ies, which ha e no been su icien ly in es iga ed o hese ma e ials. S aphylococcus au eus is one o he mos common pa hogenic bac e ia ha causes local in ec ion. 29 Bo h po ous oam i on samples and solid i on samples in he o m o pelle s wi h a ba e polyme ic and bioac i e coa ing con aining gen amicin we e p epa ed in his wo k and hen examined o deg ada ion p ope ies, as well as cy o oxici y and an ibac e ial p ope ies. The ob ained esul s demons a ed he sui abili y o he p epa ed ma e ials o po en ial use in heal h ca e, mainly due o hei sui able deg ada ion p ope ies, good biocompa - ibili y, and an ibac e ial p ope ies. Figu e 1. (a) Polyme ic and (b) bioac i e coa ing deposi ion scheme. ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27114 2. MATERIALS AND METHODS 2.1. I on Foam P epa a ion. Foam i on samples ( Fe) we e p epa ed by pou ing PUR (polyu e hane) oam (Fil en, Czech Republic) cylinde s wi h a diame e o abou 1 cm in o a suspension p epa ed by dissol ing 0.2 g o gela in (Sigma- Ald ich, USA) in 6 mL o dis illed wa e and hen adding o ca bonyl i on powde , (BASF, ype CC d50, ac ion 3.8−5.5 μm) which was used as he base ma e ial. The suspension- imp egna ed cylind ical samples we e sin e ed in an Ane a 1 ube u nace (ANETA, T encianskaTepla, Slo akia) in wo s eps. The i s s ep, a 450 °C o 120 min in an ine a mosphe e (N2) o emo e he PUR oam. The second s ep, he sin e ing o he suspension-imp egna ed cylinde s hemsel es a 1120 °C, o 60 min ook place in a educing a mosphe e o hyd ogen (hea ing a e 5 °C/min, cooling a e 4−5°C). 2.2. I on Compac P epa a ion. Compac samples (cFe) we e p epa ed om ca bonyl i on powde (CIP, BASF, ype CC d50, ac ion 3.8−5.5 μm) by cold p essing in o pelle s wi h a diame e o 12 mm a a p essu e o 600 MPa. The p essed samples we e hen sin e ed a 1120 °C in a educing a mosphe e o hyd ogen o 1 h. 2.3. Su ace Modi ica ion o he P epa ed Ma e ial. 2.3.1. Deposi ion o Polyme Coa ing on he I on Samples. The su ace o bo h he po ous oam i on samples and he compac i on samples was modi ied wi h a poly(e hylene glycol) 4000 (Sigma-Ald ich, USA) coa ing laye . An e hanol solu ion con aining 10 w % PEG was p epa ed. The samples we e i s cleaned wi h sandpape and hen ul asonically o 10 min in ace one and e hanol and hen imme sed in he PEG solu ion o 3 h a oom empe a u e and d ied o ano he 3 h a 45 °C (Figu e 1a). The samples we e ma ked as Fe-PEG and cFe-PEG. 2.3.2. Deposi ion o Bioac i e Coa ing on I on Samples. A pa o he oam and compac i on samples was modi ied wi h a polyme ic bioac i e coa ing consis ing o PEG and gen amicin (Figu e 1b). An e hanol solu ion was p epa ed con aining 10 w % PEG and 300 mg o gen amicin sul a e (cell-cul u e es ed, 590 μg o gen amicin base/mg, Sigma G-1264) o 50 mL o solu ion. The compac samples we e i s cleaned wi h sandpape and hen ul asonically o 10 min in ace one and e hanol and hen imme sed in he PEG solu ion con aining gen amicin o 3 h a oom empe a u e and d ied o ano he h ee h a 45 °C. The samples we e ma ked as Fe-PEG + Ge and cFe-PEG + Ge. 2.4. Cha ac e iza ion o Ma e ials. 2.4.1. Su ace Mo phology and Composi ion. Mac oscopic images o he p epa ed ma e ials we e aken wi h a Dino-Li e P emie AM4013MT digi al mic oscope (1.3 MPx, 20×magni ica ion). The mo phology o he p epa ed samples was s udied by scanning elec on mic oscopy (SEM) and he su ace composi ion by ene gy dispe sion analysis (EDX) (JEOL JSM-7000F, Japan wi h EDX INCA). The speci ic su ace a ea o he samples was de e mined by he low- empe a u e ni ogen adso p ion me hod, and he speci ic su ace a ea alues o he es ed samples ( alues ep esen he a e age o i e measu emen s) we e ob ained using he B unaue −Emme −Telle (BET) me hod (NOVA 1200 e Su ace A ea and Po e Size Analyze , Quan ach ome Ins umen s, London, UK). FTIR (Fou ie ans o m in a ed spec oscopy) spec a we e eco ded on an in a ed spec ome e by using he ATR (A enua ed To al Re lec ance) me hod (B uke Op ik GmbH, E lingen, Ge many). The po osi y o he p epa ed oam ma e ials was de e mined by using ImageJ so wa e. To calcula e he po osi y o he i on samples, he SEM images we e con e ed o RGB o ma and digi ized in an ImageJ Analyze . Pixel segmen a ion was hen pe o med using a h eshold o mula dis inguishing be ween black pixels (po osi y) and g ay pixels (sample), allowing he o al op ical po osi y o be quan i ied. 30 XPS (X- ay pho oelec on spec oscopy) da a we e eco ded using a The mo Scien i ic K-Alpha XPS sys em (The mo Fishe Scien i ic, UK) equipped wi h a mic o ocused monoch oma ic Al KαX- ay sou ce (1486.6 eV). A 400 μm X- ay beam a 6 mA ×12 kV was used. Spec a we e acqui ed in he cons an ene gy mode o he analyze wi h a pass ene gy o 200 eV o he su ey. Na ow egions we e collec ed wi h a pass ene gy o 50 eV, wi h an ene gy s ep size o 0.1 eV. The The mo Scien i ic Ad an age so wa e, e sion 5.9931 (The mo Fishe Scien i ic), was used o digi al acquisi ion and da a p ocessing. The su ace composi ion (a omic %) was de e mined by conside ing he in eg a ed peak a eas o he de ec ed a oms and he co esponding sensi i i y ac o s. Each spec um ep esen s he a e age o he h ee measu emen s. 2.4.2. Elec ochemical Measu emen s. The p epa ed ma e ials we e subjec ed o a dynamic deg ada ion es by means o an anodic pola iza ion me hod using an Au olab PGSTAT 302N po en ios a . A h ee-elec ode sys em was used in which he p epa ed sample was a wo king elec ode, a sil e chlo ide elec ode (Ag/AgCl/KCl (3 mol/L) was a e e ence elec ode, and a pla inum elec ode was used as an auxilia y elec ode. The po en ials we e scanned in he ange om -400 o -800 mV a a scan a e o 0.1 mV/s. Du ing he es , he samples we e imme sed in Hanks’ solu ion, which is used as a simula ed physiological en i onmen . I is a balanced sal solu ion ha mimics he ionic composi ion o human ex acellula luid and p o ides a sui able en i onmen o e alua ing ma e ial deg ada ion and biocompa ibili y (wi h composi ion: 8 g/L NaCl; 0.4 g/L KCl; 0.14 g/L CaCl2; 0.1 g/ L MgSO4·7H2O; 0.1 g/L MgCl2·6H2O; 0.06 g/L Na2HPO4· 2H2O; 0.06 g/L KH2PO4; 1 g/L Glucose; 0.35 g/L NaHCO3, and pH = 7.4 ±0.2) and empe ed a 37 ±2°C. Th ee samples we e s udied o each analysis. The co osion a e was subsequen ly calcula ed using he Ta el ex apola ion me hod acco ding o eq 1 based on ASTM G59: 31 = j d CR KEW co (1) whe e CR is he co osion a e (mm/yea ), jco is he cu en densi y (A/cm2), Kis he cons an de e mining he esul ing uni s, EW is he equi alen weigh o he ma e ial, and dis he ma e ial densi y (g/cm3). A e he measu emen , he samples we e emo ed om he Hanks’ solu ion, insed wi h e hanol, and d ied in ai . P io o he s a o he deg ada ion es s, he open ci cui po en ial (OCP) was eco ded o 60 min a e solu ion s abiliza ion. The OCP alue was used in he measu emen o elec ochemical impedance spec oscopy (EIS), which was pe o med wi h he same h ee-elec ode sys em as o he elec ochemical deg ada ion es . The samples we e imme sed in 50 mL o Hanks’ solu ion du ing he measu emen . The measu emen ook place in he equency ange o 10 mHz− 100 kHz wi h an al e na ing cu en ampli ude o 10 mV. Gen amicin elease es s we e ca ied ou using EIS and ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27115 conduc i i y measu emen s. EIS measu emen s we e ca ied ou by using Sola on Analy ical Modulab (mdl. 2100 A), wi hin he equency ange om 100 kHz o 1 Hz wi h ampli ude 10 mV a he po en ial 15 mV s e e ence elec ode. The EIS measu emen da a we e i ed and e alua ed by using he Z iew p og am. The PBS solu ion was used o EIS measu emen , and dis illed wa e was used o conduc i i y measu emen s. These measu emen s we e pe o med ia a WTW Inolab conduc i i y me e Le el 1. 2.4.3. Imme sion Deg ada ion Tes s. The imme sion co osion es was also pe o med in Hanks’ solu ion. P io o he s a o he es , he samples we e weighed (mi) and hen ul asonically cleaned in ace one and e hanol o 10 min. Subsequen ly, he h ee se s o es samples we e imme sed in Hanks’ solu ion. The i s se o samples was subjec ed o an imme sion co osion es o 4 weeks, he second o 8 weeks, and he hi d o 12 weeks a 37 °C. Th ee samples om each species we e s udied in each se . A he end o he es , he samples we e ul asonically cleaned in ace one and e hanol o 10 min o emo e he excess co osion p oduc s, hen ai -d ied and weighed (m ). The co osion a e was de e mined om he change in weigh acco ding o he eq 2 based on ASTM G31 s anda d: 32 = m m K A d CR ( ) i (2) whe e CR is he co osion a e, m is he mass o he sample a he end o he es , miis he mass o he sample a he beginning o he es , Kis cons an (87600), Ais he su ace a ea o he sample, is he exposu e ime, and dis he ma e ial densi y. 2.4.4. Cy o oxici y Tes . The sample oxici y es ing was pe o med in i o acco ding o STN ISO 10993-5 no m 33 a 37 °C. Samples o Fe, cFe, Fe-PEG, cFe-PEG, Fe-PEG + Ge, cFe-PEG + Ge, and s ainless s eel (SS) we e s e ilized by UV and placed in polyp opylene (PP) cen i uge ubes, whe e 2 mL o he cul u e medium consis ing o Dulbecco’s modi ied Eagle’s medium (DMEM) wi h 10% e al bo ine se um (FBS) and 1% an ibio ic solu ion (ATB) was added. To ob ain he ex ac s, he samples we e imme sed in he cul u e medium o wo di e en ime in e als, 4 and 24 h. Subsequen ly, samples we e aken om he ubes and he ob ained ex ac s we e cen i uged o 5 min a 10,000 pm. The cell popula ion was de e mined in a Bu ke chambe p io o he expe imen s. Cen i uged ex ac s we e sub- sequen ly used o de e mine he in i o cy o oxici y. Human de mal ib oblas (Human De mal Fib oblas s, HDFa; Sigma- Ald ich) cells we e placed in a 96-well pla e (G ade B and cul u e mic opla e, adhe en cells); 100 μL o cul u e medium was added o each well o he pla e so ha he e was 104HDFa in each cell. Cul i a ion un il he o ma ion o monolaye s ook place in an incuba o (37 °C, 95% humidi y, and 5% CO2). A e 24 h o incuba ion, he cul u e medium om each well was emo ed and subsequen ly, p epa ed ex ac s we e added o he wells wi h seeded cells and we e le o incuba ion o 4 h. A e incuba ion, he ex ac om each well was pipe ed o and he cy o oxici y was de e mined by MTS p oli e a ion assay (CellTi e 96 AQueous one solu ion cell p oli e a ion assay, P omega, USA). 100 μL o MTS eagen was placed in each well o he pla e, which was placed in an incuba o a 37 °C o 4 h. A e wa d, he abso bance o o mazan was de e mined a 490 nm using UV VIS spec opho ome e (Shimadzu), and hen cell iabili y was calcula ed using eq 3: = ×V(%) OD OD 100% NC (3) whe e OD is he op ical densi y o he i on samples and ODNC is he op ical densi y o he nega i e con ol. The expe imen was epea ed h ee imes o each sample using wells wi hou ex ac s as a nega i e con ol. 2.4.5. An ibac e ial Ac i i y Tes . The an ibac e ial ac i i y o Fe-PEG and Fe-PEG + Ge was es ed agains bac e ial s ains o Esche ichia coli CCM 3954 and S aphylococcus au eus CCM 4223 (Czech Collec ion o Mic oo ganisms, B no). 2.4.5.1. Disc Di usion Me hod. The bac e ial s ains es ed o de e mine he an ibac e ial ac i i y o Fe-PEG and Fe-PEG + Ge we e cul i a ed o 18 h. Subsequen ly, he suspensions we e p epa ed in a s e ile physiological solu ion and adjus ed o a alue o 0.5 on he McFa land scale. The hus-p epa ed suspensions we e inocula ed on Muelle −Hin on aga (MHA) in a olume o 100 μL. Consequen ly, 10 μL o PEG and PEG + Ge we e added on pape discs wi h a diame e o 6 mm in a concen a ion ange om 6 o 0.047 mg/mL. An ibac e ial ac i i y was e alua ed by measu ing he diame e o he inhibi ion zone in millime e s. An an ibio ic disc wi h gen amicin (10 μg) was used as a con ol. Figu e 2. SEM images o oam and compac i on-based samples: (a) Fe, (b) Fe-PEG, (c) Fe-PEG + Ge, (d) cFe, (e) cFe-PEG, and ( ) cFe-PEG + Ge. ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27116 2.4.5.2. Spec opho ome ic Tes . The an ibac e ial ac i i y o he Fe-PEG + Ge solu ion was de e mined spec opho o- me ically by measu ing he abso bance. The PEG + Ge solu ion was dilu ed in BHI (B ain-Hea In usion) b o h in 96-well pla es in a concen a ion ange o 150−4.7 μg/mL. The es ed bac e ial s ains we e cul i a ed o 18 h, and he p epa ed suspensions in a s e ile physiological solu ion we e adjus ed o a alue o 1.0 on he McFa land scale. Subsequen ly, he bac e ial suspensions we e added o he dilu ed PEG + Ge solu ion in a a io o 1:1. A e 24 h o incuba ion a 37 °C, he an ibac e ial ac i i y was spec opho- ome ically de e mined by measu ing he abso bance a a wa eleng h o 600 nm using a Bio ek Syne gy 2 de ice. BHI b o h wi h he es ed bac e ial s ains was used as a con ol. The esul s we e e alua ed by using he Dunne es in he s a is ical p og am P ism 8.3.0. 3. RESULTS AND DISCUSSION 3.1. Mo phology and Su ace Composi ion. SEM images o he su ace o he p epa ed oam and compac (Figu e 2) ma e ials we e aken as u he con i ma ion o he p esence o he polyme ic coa ing. Bo h he mic opo es (wi h a size om 0.5 o 5 μm) and mac opo es (wi h a size om 450 o 1500 μm) we e p esen in he p epa ed oam samples. In he case o coa ed i on oams, he mac opo e size dec eased, and he walls hickened as compa ed o he uncoa ed oam samples. As a esul o he coa ing deposi ion, he cells we e pa ially o wholly illed wi h polyme , and he mac opo es we e educed o comple ely closed. Mo eo e , he deposi ion o he PEG coa ing caused he mic opo e closu e, smoo hing ou he s uc u e and c ea ing a glossie and smoo he su ace o he ma e ial. These changes in he su ace o he coa ed samples a e well obse able om he SEM images depic ed in Figu e 2b,c. The p esence o sc a ches on he su ace o he uncoa ed compac samples (Figu e 2d) was obse ed due o he cleaning o he ma e ial wi h sandpape s p io o coa ing. Applica ion o he pu e PEG coa ing as well as he coa ing con aining gen amicin esul ed in he smoo hing o he su ace o he ma e ial (Figu e 2e, ). The po osi y o he p epa ed oam samples was de e mined o be 56.23% (Figu e 3a), 44.97% (Figu e 3b), and 41.32% (Figu e 3c) o he Fe, Fe-PEG, and Fe-PEG + Ge samples, espec i ely. Black a eas in Figu e 3 show he po es ha a e p esen . To examine he su ace p ope ies o he p epa ed oam samples, we de e mined he speci ic su ace a ea alues (SBET) o he samples we e de e mined. The esul s a e shown in Table 1. The speci ic su ace a ea alues o he Fe-PEG and Fe-PEG + Ge samples we e lowe by almos hal compa ed o he uncoa ed Fe, which is ela ed o he smoo hing o he su ace and he educ ion o he po e size o he polyme - coa ed samples. The su ace a eas o he compac samples we e de e mined by geome ic calcula ion. The a e age su ace a ea o he compac samples was 3.18 ×10−4m2which co esponds o a speci ic su ace a ea o app oxima ely 1.59 ×10−4m2/g. The p esence o a polyme ic PEG coa ing laye on he su ace o he coa ed samples was con i med by he su ace EDX analysis based on he p esence o oxygen and ca bon on he su ace o he analyzed cFe-PEG, Fe-PEG, cFe-PEG + Ge, and Fe-PEG + Ge samples, which we e no obse ed in he case o pu e i on. Ni ogen and sul u we e no de ec ed on he su ace o any oam samples due o hei low con en . In he case o he compac cFe-PEG + Ge sample, he p e-p esence o sul u was de ec ed o con i m he gen amicin sul a e on he su ace. The a e age alues o he con en o indi idual elemen s on he su ace a e lis ed in Table 2. In he case o he Fe-PEG + Ge sample, he p esence o i on was obse ed on he su ace, which was caused by he une en dis ibu ion o he coa ing on he sample su ace and he ips o Fe nodes p o uding om he coa ing laye . In he case o uncoa ed i on samples ( Fe and cFe), only Fe was obse ed on he su ace. The p esence o he polyme coa ing was also con i med by in a ed spec oscopy. Figu e 4a shows he in a ed spec a o he pu e PEG, Ge, Fe, Fe-PEG, and Fe-PEG + Ge samples. In he in a ed spec um o pu e PEG, unc ional g oup ib a ions we e iden i ied as ollows: alence ib a ions o he −OH g oup a 3400 cm−1, asymme ic and symme ic alence ib a ions o he −CH2g oup a 2869 cm−1, alence ib a ions o he −CO g oup a 1099 cm−1and de o ma ion ib a ions o CH g oups a 960 and 840 cm−1. The abso p ion bands a 1461, 1359, and 1280 cm−1 u he cha ac e ize he de o ma ion ib a ions o CH2g oups. The p esence o a iple peak o alence ib a ions C−C and C−O in he ange om 1000 o 1200 cm−1is e idence o he exis ence o a c ys alline phase and was ound in he spec um o pu e PEG as well as in Fe-PEG and Fe-PEG −Ge samples. 34 In he in a ed spec um o pu e gen amicin, unc ional g oup ib a ions we e iden i ied as ollows: he amide (N−H) bending ib a ions o p ima y a oma ic amines a 1620 and 1524 cm−1and he S−O bending ib a ion and S−O s e ch a 600 and 1040 cm−1. 35 No gen amicin peaks we e de ec ed in he spec um o he Fe-PEG −Ge sample due o he low con en o gen amicin in he polyme coa ing; he e o e, PEG peaks p edomina ed in he sample. Figu e 3. Po osi y de e mina ion o oam (a) Fe, (b) Fe-PEG, and (c) Fe-PEG + Ge samples using ImageJ so wa e. Table 1. Speci ic Su ace A ea Values o Po ous Foam Samples sample SBET (m2/g) Fe 0.34 Fe-PEG 0.17 Fe-PEG + Ge 0.21 ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27117 Mo eo e , XPS analysis was pe o med o u he examine he chemical s a e o elemen s p esen a he sample su ace. In he case o he Fe sample, he p esence o peaks co esponding o oxygen on he su ace due o he o ma ion o i on oxides ( he main Fe 2p signal a 710.9 eV co esponding o i on oxides, Figu e 4b, Table 3) on he su ace o he sample was obse ed. In he Fe-PEG sample, he su ace was almos comple ely co e ed wi h polyme ; he e o e, only peaks co esponding o Table 2. Su ace Composi ion o Fe, Fe-PEG, and Fe-PEG −Ge Samples De e mined by EDX Analysis Fe C O S w % a % w % a % w % a % w % a % oam samples Fe 100 100 Fe-PEG 62.27 68.73 37.73 31.27 Fe-PEG + Ge 16.56 4.44 56.04 69.90 27.40 25.66 compac samples cFe 100 100 cFe-PEG 64.08 70.38 35.92 29.62 cFe-PEG + Ge 64.17 70.46 35.83 29.54 0.41 0.33 Figu e 4. (a) In a ed spec um o he p epa ed s udied ma e ials −Fe, Fe-PEG, Fe-PEG + Ge, pu e polye hylene glycol (PEG), and gen amicin sul a e (Ge), (b) su ey XPS spec a o p epa ed Fe, Fe-PEG, and Fe-PEG + Ge samples, and (c) su ey XPS spec a o p epa ed Fe, Fe-PEG, and Fe-PEG + Ge samples. Table 3. Appa en Su ace Chemical Composi ion De e mined by XPS sample su ace chemical composi ion (a %) C 1s O 1s Fe 2p N 1s S 2p Fe 46.4 41.2 10.2 2.3 Fe-PEG 78.5 21.5 Fe-PEG + Ge 68.1 30.9 0.7 0.3 ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27118 oxygen and ca bon om he PEG coa ing we e obse ed (see Figu e 4c, C 1s a ca. 286.0 eV con i ms C−O om PEG). Based on he S 2p and N 1s peaks, he p esence o gen amicin in he Fe-PEG + Ge sample was con i med. Some ni ogen was also obse ed in he case o he Fe sample, which is mainly a ca. 400.0 eV, co esponding o he C−N g oup. This p obably comes om some con amina ion o he Fe su ace. In he case o he Fe-PEG + Ge sample, N 1s is mainly a 401.1 eV, co esponding o −NH3+and S 2p is a ca. 168.2 eV, co esponding o sul a e. This clea ly con i ms he p esence o cha ged gen amicin sul a e on he su ace. Fe-PEG + Ge is ully coa ed by PEG (68.1 a % o ca bon wi h main C−O signal a 286.0 eV, Table 3,Figu e 4c). A ce ain amoun o gen amicin is bound o he su ace o his laye . Simila esul s we e obse ed in s udies whe e he p esence o su ace-bound d ugs (gen amicin) was obse ed on mic osphe es p oduced using polylac ic acid (PLLA) and copolyme o lac ic acid and glycolic acid (PLGA). 36,37 3.2. Co osion Beha io . 3.2.1. Elec ochemical Co o- sion Beha io . The OCP po en ial was egis e ed o 60 min. A e abou 40 min, he OCP alues s abilized in he ange om −0.54 o −0.62 V o each po ous oam sample (Figu e 5a) and in he ange om −0.48 o −0.57 V o each compac sample (Figu e 5b). The lowes OCP alues we e obse ed o he Fe-PEG, cFe-PEG, Fe-PEG + Ge, and cFe-PEG + Ge samples. This indica es an inc eased endency o co osion in he coa ed samples. To de e mine he co osion a e (CR), dynamic pola iza ion es s we e pe o med in Hanks’ solu ion a 37 °C. Table 4 shows he alues o co osion po en ial (Eco ), co osion cu en densi y (jco ), and CR alues de e mined by he Ta el ex apola ion me hod om he po en iodynamic pola iza ion cu es (Figu e 5c, d). A po en ial shi o a mo e nega i e alue was obse ed o bo h compac and oam samples wi h he PEG coa ing and he PEG coa ing con aining gen amicin, indica ing a highe endency o co osion compa ed wi h he uncoa ed samples. Figu e 5. Time dependence o he OCP o (a) oam i on-based samples, (b) compac i on-based samples, and po en iodynamic pola iza ion cu es o (c) oam, and (d) compac samples Fe, Fe-PEG, and Fe-PEG + Ge in Hanks’ solu ion. Table 4. Values o jco ,Eco and Co osion Ra es o he Fe, Fe-PEG, and Fe-PEG −Ge Samples Eco (mV) jco (μA·m−2) CR(mmpy) oam samples Fe −558 28.685 0.333 Fe-PEG −604 62.555 0.727 Fe-PEG + Ge −610 64.750 0.752 compac samples cFe −444 25.319 0.294 cFe-PEG −532 47.543 0.552 cFe-PEG + Ge −548 55.264 0.642 ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27119 The inc ease in he co osion a e o he coa ed samples as compa ed o he uncoa ed sample can be a ibu ed o he sligh acidi y o he co osion medium in he icini y o he su ace o he coa ed samples due o he oxida i e deg ada ion o PEG. The deg ada ion o PEG begins wi h oxida ion o he e minal OH g oup and spli ing o he hyd ogen a om, which leads o a dec ease in he pH o he solu ion. The change in pH is also caused by he in e ac ion be ween he polyme and wa e . A local educ ion o he pH alue nea he su ace o he coa ed samples subsequen ly leads o inc eased solubili y o co osion p oduc s and he o ma ion o a less compac and dense passi e laye , which accele a es hei deg ada ion. 38 The acidic en i onmen leads o inc eased p o on educ ion a he ca hode, which can cause highe co osion cu en densi y and hus a highe co osion a e. 39,40 When i on-based subs a es wi h polye hylene glycol (PEG) coa ing a e exposed o Hanks’ solu ion, he co osion a e inc eases due o he enhanced oxida ion a e o i on caused by he in e ac ion be ween he hyd ophilic polyme laye and he i on su ace. 41,42 Howe e , i is also possible o in luence he ime o i s deg ada ion by he hickness o he polyme laye and hus adjus he p ope ies o he bioma e ial acco ding o he equi emen s o he gi en applica ion. Nyquis diag ams o he p epa ed samples ob ained be o e and a e 60 min imme sion in Hanks’ solu ion a e shown in Figu e 6a,b. The diag ams demons a e ha he i on-based samples exhibi wo ypes o loops: a capaci i e loop in he high and medium equency ange and he induc ion loop in he low- equency ange. The capaci i e loop is ela ed o cha ge ans e , and he induc i e loop is caused by he dissolu ion o i on. 43 Fo he oam as well as he compac samples, he high- equency capaci i e loop was desc ibed using he capaci ance and cha ge ans e esis ance, which cha ac e ized he bulk laye o he co osion p oduc s. 44 The occu ence o he semici cle in he low- equency ange e lec s he su ace inhomogenei y o he samples p epa ed by he powde me allu gy me hod. The semici cle diame e s o he cFe and Fe samples a e la ge han hose o he cFe-PEG, Fe-PEG, cFe-PEG + Ge, and Fe-PEG + Ge samples, indica ing highe cha ge ans e esis ance. The Nyquis diag ams o he uncoa ed and coa ed samples we e modeled using he equi alen ci cui shown in Figu e 6c. In his ci cui , Rs ep esen s he solu ion esis ance, Rc ep esen s he polyme laye esis ance, Rc ep esen s he cha ge ans e esis ance, and CPE a e elemen s o a cons an phase. The Rc alues, which ep esen he pola iza ion esis ance, we e calcula ed o all samples as he di e ence in impedance a lowe and highe equencies (Table 5). The alues o oam and he compac samples wi h he PEG coa ing laye as well as o oam and he solid samples con aining gen amicin we e lowe han hose o samples wi hou he polyme laye indica ing lowe esis ance o co osion, which con i ms he same end as po en iodynamic pola iza ion measu emen s Highe Rc alues o uncoa ed cFe and Fe samples indica e he o ma ion o a passi a ion laye o deg ada ion p oduc s, such as i on oxides, i on hyd oxides, and ca bona es, while Figu e 6. Nyquis diag am o (a) oam, (b) compac i on-based Fe, Fe-PEG and Fe-PEG + Ge samples be o e co osion and (c) equi alen ci cui −Rs−solu ion esis ance, Rc−polyme laye esis ance, Rc −cha ge ans e esis ance, CPE −elemen s o a cons an phase. Table 5. Impedance Pa ame e Rc o Foam and Compac Fe, Fe-PEG, and Fe-PEG + Ge Samples Rc [Ω·m−2] oam samples compac samples Fe 168.93 312.74 Fe-PEG 114.90 254.41 Fe-PEG + Ge 100.07 235.21 ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27120 lowe alues ob ained o he coa ed compac (cFe-PEG, cFe- PEG + Ge) and oam ( Fe-PEG, Fe-PEG + Ge) samples indica e he desi ed highe co osion a e. The disc epancy be ween he inc ease in he Rc alue o he oam Fe-PEG + Ge and compac cFe-PEG + Ge samples compa ed wi h he Fe and cFe samples may be ela ed o he complex na u e o he laye o med and he pa ial b eakdown o he su ace ilm in some a eas due o he addi ion o he an ibio ic. 3.2.2. Imme sion Co osion Beha io . The s a ic imme - sion deg ada ion me hod was also used o de e mine he a e o deg ada ion. The mac oscopic images o he su ace o he ma e ials a e imme sion in Hanks’ solu ion o 4, 8, and 12 weeks a e shown in Figu e 7a,b. A e 4 weeks o con inuous imme sion co osion es ing, i was s ill possible o obse e se e al unco oded si es on he su aces o co oding samples. A hin laye o o ange and b own co osion p oduc s was isible on he su ace o he Fe sample (Figu e 7a). In he case o he Fe-PEG and Fe-PEG + Ge samples, a mo e p onounced co e age o he samples wi h co osion p oduc s was obse ed. A e 12 weeks o co osion, he oam samples we e ela i ely agile; he walls o he samples we e signi ican ly damaged, and he su aces o he samples we e almos comple ely co e ed wi h a laye o co osion p oduc s. The same end was obse ed o compac samples (Figu e 7b). Howe e , e en a e 12 weeks o co osion, he compac samples kep hei shape ela i ely in ac , which is due o he signi ican ly smalle po osi y and se e al imes smalle su ace a ea on which he co osion ook place. The deg ada ion a e o o hopedic implan s depends on he speci ic applica ion and equi emen s o implan . Fo small inju ies, implan s wi h accele a ed deg ada ion may lead o p ema u e loss o mechanical suppo . 45 Con e sely, in cases equi ing medium-speed deg ada ion, he use o implan s wi h con olled deg ada ion a es can ensu e p ope healing wi hou comp omising s uc u al in eg i y. 46 The biodeg ada ion a e o he samples was de e mined by measu ing he weigh loss a e imme sion in Hanks’ solu ion (Figu e 7c,d). The calcula ed co osion a es based on he con inuous imme sion es o bo h p essed and oamed samples a e 4, 8, and 12 weeks a e shown in Table 6. A e 12 weeks o he imme sion es , he deg ada ion a e o pu e Fe oam was 0.025 mm/yea ; he deg ada ion a e o Fe Figu e 7. Mac oscopic images o i on-based samples a e 4, 8 and 12 week imme sion in Hanks’ solu ion a magni ica ions o 20×, (a) oam, (b) compac samples and mass losses du ing imme sion in Hanks’ solu ion o 4, 8, and 12 weeks o i on-based (c) oam ( Fe, Fe-PEG, Fe-PEG + Fe) (d) compac (cFe, cFe-PEG, cFe-PEG + Ge) samples. ACS Omega h p://pubs.acs.o g/jou nal/acsod A icle h ps://doi.o g/10.1021/acsomega.4c01002 ACS Omega 2024, 9, 27113−27126 27121