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*
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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
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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.
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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
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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.
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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
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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
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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
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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
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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.
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