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

Ma e ials 2022,15, 7487 6 o 13
and le o 24 h a 37
◦
C. A e 2 h a 37
◦
C, samples we e washed h ee imes in o de o
elimina e he non-a ached bac e ia, and hFFs in modi ied DMEM (DMEM wi h 2% BHI) a
2×104cells/sample we e seeded and incuba ed o 24 h.
The esul s we e s a is ically s udied by S uden ’s - es s, one-way ANOVA ables
and Tu key’s mul iple compa ison es s. Using his me hod can e alua e any s a is ically
signi ican di e ences be ween he sample g oups. The signi icance di e ences we e when
p< 0.05. The s a is ical s udy was ealized by Mini abTM so wa e (Mini ab elease 13.0,
Mini ab Inc., S a e College, PA, USA).
3. Resul s
Roughness s udies we e ca ied ou on he mini-implan s since, as is well known,
oughness is a pa ame e ha will a ec bac e ial coloniza ion. The esul s o he o igi-
nal i anium (Ti), i anium ac i a ed by a gon plasma (PA) and hose ea ed wi h PEG
polyme iza ion plasma is shown in Table 2. F om hese esul s, no s a is ically signi ican
di e ences can be obse ed in any case. The ea men does no a ec he opog aphy o
he samples.
Table 2. Roughness o he di e en mini-implan s.
Sample Ra(µm) Pc(cm−1)
Ti 0.33 ±0.10 150.9 ±69
PA 0.35 ±0.20 153.4 ±56
PEG100-30 0.36 ±0.30 152.8 ±59
PEG100-60 0.33 ±0.21 146.9 ±60
PEG150-30 0.43 ±0.12 150.9 ±69
PEG150-60 0.37 ±0.09 150.9 ±69
PEG200-30 0.39 ±0.09 150.9 ±69
PEG200-60 0.32 ±0.12 150.9 ±69
We abili y inc eased wi h he plasma ac i a ion, especially wi h a gon (PA) ea men s.
This was ealized by means o a gon a a 100 W peak powe om 64.2
±
5.5
◦
o he as-
ecei ed i anium o 5.2
±
1.2
◦
ea ed wi h a gon, showing an impo an supe -hyd ophilic
cha ac e . The con ac angles o he di e en ea men s wi h PEG a e shown in Table 3.
Table 3.
Con ac angles o he di e en samples wi h PEG. As e isks indica e he s a is ical di e -
ence signi icances.
Sample Con ac Angle (◦)
PEG100-30 12.3 ±0.9 *
PEG100-60 13.2 ±1.3 *
PEG150-30 18.2 ±1.2 **
PEG150-60 19.1 ±0.8 **
PEG200-30 25.0 ±2.2 ***
PEG200-60 25.2 ±2.9 ***
S a is ically signi ican di e ences in he con ac angle can be obse ed wi h he
di e en peak powe alues applied, bu no di e ences in ea men imes a e obse ed o
each peak powe applied.
The a omic concen a ion o he elemen s in he ou e su ace was eco ded by XPS
and is summa ized in Table 4.
Cy ocompa ibili y esul s demons a ed no dec ease a any dilu ion when cul u ed
wi h ib oblas s and os eoblas s (Figu es 6and 7, espec i ely). All o he s udied su aces
and he plasma polyme iza ion condi ions had cy ocompa ibili y a ios o e 70%. Fo bo h
esul s, he e a e no signi ican s a is ical di e ences. The excellen biocompa ibili y o he
ea men s can be concluded.
Ma e ials 2022,15, 7487 7 o 13
Table 4.
A omic concen a ion (in %) o he ca bon, oxygen and i anium amoun p esen on he Ti,
and PA.
Sample O 1s C 1s Ti 2p
Ti 55 ±1 24 ±1 20 ±1
PA 63 ±2 10 ±1 26 ±1
PEG100-30 45 ±2 41 ±1 2 ±1
PEG100-60 54 ±3 37 ±2 5 ±1
PEG150-30 42 ±1 47 ±1 8 ±1
PEG150-60 45 ±1 52 ±1 8 ±1
PEG200-30 48 ±2 44 ±2 7 ±1
PEG200-60 40 ±1 52 ±3 6 ±1
Ma e ials 2022, 15, 7487 8 o 14
Figu e 6. Cell iabili y o he hFFs o he di e en ea men s and di e en dilu ions.
Figu e 7. Cell iabili y o he SAOS-2 o he di e en ea men s and di e en dilu ions.
Cell adhesion assays wi h hFFs (Figu e 8) showed no di e ence be ween Ti, PA and
PEG samples, whe eas, o SAOS-2 (Figu e 9), a sligh ly dec ease was measu ed when he
samples we e polyme ized.
Figu e 6. Cell iabili y o he hFFs o he di e en ea men s and di e en dilu ions.
Ma e ials 2022, 15, 7487 8 o 14
Figu e 6. Cell iabili y o he hFFs o he di e en ea men s and di e en dilu ions.
Figu e 7. Cell iabili y o he SAOS-2 o he di e en ea men s and di e en dilu ions.
Cell adhesion assays wi h hFFs (Figu e 8) showed no di e ence be ween Ti, PA and
PEG samples, whe eas, o SAOS-2 (Figu e 9), a sligh ly dec ease was measu ed when he
samples we e polyme ized.
Figu e 7. Cell iabili y o he SAOS-2 o he di e en ea men s and di e en dilu ions.
Ma e ials 2022,15, 7487 8 o 13
Cell adhesion assays wi h hFFs (Figu e 8) showed no di e ence be ween Ti, PA and
PEG samples, whe eas, o SAOS-2 (Figu e 9), a sligh ly dec ease was measu ed when he
samples we e polyme ized.
Ma e ials 2022, 15, 7487 9 o 14
Figu e 8. Cell adhesion o hFFs on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol
ha e no s a is ically signi ican di e ence be ween hem (p < 0.05).
Figu e 9. Cell adhesion o SaOS-2 on he Ti, PA and PEG samples. Ba s indica ed wi h he same
symbol ha e no s a is ically signi ican di e ence be ween hem (p < 0.05).
Bac e ial adhesion es s showed a dec eased bac e ial adhesion o all PEG samples,
ei he o he Spec ococcus sanguinis and he Lac obacillus sali a ius (Figu es 10 and 11,
espec i ely). Ti samples and plasma-ac i a ed samples (PA) we e used as con ols. An
inc eased bac e ial adhesion was obse ed o he PA sample compa ed o Ti.
Figu e 8.
Cell adhesion o hFFs on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol
ha e no s a is ically signi ican di e ence be ween hem (p< 0.05).
Ma e ials 2022, 15, 7487 9 o 14
Figu e 8. Cell adhesion o hFFs on he Ti, PA and PEG samples. Ba s indica ed wi h he same symbol
ha e no s a is ically signi ican di e ence be ween hem (p < 0.05).
Figu e 9. Cell adhesion o SaOS-2 on he Ti, PA and PEG samples. Ba s indica ed wi h he same
symbol ha e no s a is ically signi ican di e ence be ween hem (p < 0.05).
Bac e ial adhesion es s showed a dec eased bac e ial adhesion o all PEG samples,
ei he o he Spec ococcus sanguinis and he Lac obacillus sali a ius (Figu es 10 and 11,
espec i ely). Ti samples and plasma-ac i a ed samples (PA) we e used as con ols. An
inc eased bac e ial adhesion was obse ed o he PA sample compa ed o Ti.
Figu e 9.
Cell adhesion o SaOS-2 on he Ti, PA and PEG samples. Ba s indica ed wi h he same
symbol ha e no s a is ically signi ican di e ence be ween hem (p< 0.05).
Bac e ial adhesion es s showed a dec eased bac e ial adhesion o all PEG samples,
ei he o he Spec ococcus sanguinis and he Lac obacillus sali a ius (Figu es 10 and 11,
espec i ely). Ti samples and plasma-ac i a ed samples (PA) we e used as con ols. An
inc eased bac e ial adhesion was obse ed o he PA sample compa ed o Ti.
Ma e ials 2022,15, 7487 9 o 13
Ma e ials 2022, 15, 7487 10 o 14
Figu e 10. Bac e ial adhesion on Ti, PA and PEG samples o Spec ococcus sanguinis. As e isks
indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05).
Figu e 11. Bac e ial adhesion on Ti, PA and PEG samples o Lac obacillus Sali a ius. As e isks
indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05).
4. Discussion
In his s udy, we aimed o es i polye hylene glycol (PEG) coa ing would
signi ican ly p e en he o ma ion o bac e ial bio ilm on he su ace o i anium mini-
implan s. The oughness esul s show ha he oughness is no a ec ed by he plasma
ea men s. This ac is impo an since i is well known ha oughness a ec s
Figu e 10.
Bac e ial adhesion on Ti, PA and PEG samples o Spec ococcus sanguinis. As e isks
indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p> 0.05).
Ma e ials 2022, 15, 7487 10 o 14
Figu e 10. Bac e ial adhesion on Ti, PA and PEG samples o Spec ococcus sanguinis. As e isks
indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05).
Figu e 11. Bac e ial adhesion on Ti, PA and PEG samples o Lac obacillus Sali a ius. As e isks
indica ed wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p > 0.05).
4. Discussion
In his s udy, we aimed o es i polye hylene glycol (PEG) coa ing would
signi ican ly p e en he o ma ion o bac e ial bio ilm on he su ace o i anium mini-
implan s. The oughness esul s show ha he oughness is no a ec ed by he plasma
ea men s. This ac is impo an since i is well known ha oughness a ec s
Figu e 11.
Bac e ial adhesion on Ti, PA and PEG samples o Lac obacillus Sali a ius. As e isks indica ed
wi h he same symbol ha e no s a is ically signi ican di e ence be ween hem (p> 0.05).
4. Discussion
In his s udy, we aimed o es i polye hylene glycol (PEG) coa ing would signi ican ly
p e en he o ma ion o bac e ial bio ilm on he su ace o i anium mini-implan s. The
oughness esul s show ha he oughness is no a ec ed by he plasma ea men s. This
ac is impo an since i is well known ha oughness a ec s osseoin eg a ion le els,
as well as bac e ial p oli e a ion and adhesion [
40
–
44
]. These esul s make i possible
o op imize he ea men s o ob ain he op imum oughness, knowing ha subsequen
plasma ea men s will no a y his impo an cha ac e is ic.
The we abili y esul s indica e a e y signi ican a ia ion in he hyd ophilic cha ac e
when we ac i a e he su ace wi h a gon. The a ia ion in he con ac angle om 64
◦
o
5
◦
makes he su ace supe -hyd ophilic. Rega ding he su ace ea men s wi h PEG, i