pha maceu ics
A icle
An ioxidan PLA Composi es Con aining Lignin o
3D P in ing Applica ions: A Po en ial Ma e ial o
Heal hca e Applica ions
Juan Domínguez-Robles, Niamh K. Ma in, Mun Leon Fong , Sa ah A. S ewa ,
Nicola J. I win , Ma ía Isabel Rial-He mida , Ryan F. Donnelly and Eneko La añe a *
School o Pha macy, Queen’s Uni e si y Bel as , 97 Lisbu n Road, Bel as BT9 7BL, UK;
[email p o ec ed] (J.D.-R.); [email p o ec ed] (N.K.M.); [email p o ec ed] (M.L.F.)
[email p o ec ed] (S.A.S.); [email p o ec ed] (N.J.I.); [email p o ec ed] (M.I.R.-H.);
[email p o ec ed] (R.F.D.)
*Co espondence: [email p o ec ed]; Tel.: +44-(0)28-9097-2360
Recei ed: 7 Ma ch 2019; Accep ed: 2 Ap il 2019; Published: 4 Ap il 2019


Abs ac :
Lignin (LIG) is a na u al biopolyme wi h well-known an ioxidan capabili ies. Acco dingly,
in he p esen wo k, a me hod o combine LIG wi h poly(lac ic acid) (PLA) o used ilamen ab ica ion
applica ions (FFF) is p oposed. Fo his pu pose, PLA pelle s we e success ully coa ed wi h LIG powde
and a biocompa ible oil (cas o oil). The esul ing pelle s we e placed in o an ex ude a 200
◦
C.
The esul ing PLA ilamen s con ained LIG loadings anging om 0% o 3% (w/w). The ob ained
ilamen s we e success ully used o FFF applica ions. The LIG con en a ec ed he mechanical and
su ace p ope ies o he o e all ma e ial. The inclusion o LIG yielded ma e ials wi h lowe esis ance
o ac u e and highe we abili ies. Mo eo e , he esul ing 3D p in ed ma e ials showed an ioxidan
capabili ies. By using he 2,2-diphenyl-1-pic ylhyd azyl (DPPH) me hod, he ma e ials we e capable o
educing he concen a ion o his compound up o ca. 80% in 5 h. This adical sca enging ac i i y could
be po en ially bene icial o heal hca e applica ions, especially o wound ca e. Acco dingly, PLA/LIG
we e used o design meshes wi h di e en designs o wound d essing pu poses. A wound healing
model compound, cu cumin (CUR), was applied in he su ace o he mesh and i s di usion was s udied.
I was obse ed ha he dimensions o he meshes a ec ed he pe mea ion a e o CUR. Acco dingly,
he design o he mesh could be modi ied acco ding o he pa ien ’s needs.
Keywo ds: 3D p in ing; used ilamen ab ica ion; lignin; an ioxidan ma e ials; wound d essing
1. In oduc ion
The in e es in addi i e manu ac u ing (commonly known as 3D p in ing) o biomedical
applica ions has inc eased signi ican ly du ing he las decade [
1
]. Among all he di e en ypes o 3D
p in ing, used ilamen ab ica ion (FFF) is he mos commonly used [
2
]. In FFF, a polyme ilamen is
hea ed and ex uded h ough a small nozzle and subsequen ly solidi ied on a build pla e [
3
]. FFF gained
popula i y quickly a e he RepRap p ojec in 2005 [
4
]. This p ojec was ocused on de eloping low
cos do-i you sel FFF p in e s [
4
]. Acco dingly, di e en biomedical and pha maceu ical applica ions
o addi i e manu ac u ing ha e been desc ibed du ing he las decade [
1
,
5
,
6
]. These applica ions
include d ug deli e y sys ems, p os he ics o implan able de ices [
1
,
5
–
7
]. The mos common ma e ial
used o FFF is poly(lac ic acid) (PLA). This is due o i s p ocessabili y o ex usion applica ions, high
mechanical s eng h and low coe icien o he mal expansion [
8
]. Mo eo e , PLA is a enewable,
biodeg adable and biocompa ible polyme [
7
,
9
]. These p ope ies make his ma e ial ideal o
pha maceu ical and biomedical applica ions.
Pha maceu ics 2019,11, 165; doi:10.3390/pha maceu ics11040165 www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2019,11, 165 2 o 14
To p o ide ex a ea u es o PLA, di e en s a egies ha e been ollowed. The combina ion o
PLA wi h o he molecules ha can gi e added alue o he inal composi e ma e ial o heal hca e
applica ions ha e been explo ed in he pas [
10
]. One o he po en ial scena ios is he inco po a ion
o molecules wi h an ioxidan p ope ies o PLA. The un es ained p oduc ion o ee adicals and
eac i e oxygen species is linked wi h he onse o diseases such as heuma oid a h i is, a he oscle osis
o cance [
11
]. Acco dingly, he de elopmen o an ioxidan compounds/ma e ials can con ibu e
o educe he concen a ion o hese compounds [
12
]. Mo eo e , i has been shown ha he excess
o eac i e oxygen species p e en s wound healing [
13
,
14
]. Acco dingly, an ioxidan s ha e been
p oposed as a way o con ol oxida i e s ess in wounds o accele a e hei healing.
The use o an ioxidan compounds o 3D p in ing applica ions has no been ex ensi ely explo ed
in he pas . Van Li h e al. p oposed he use o s e eoli hog aphy (an al e na i e o FFF 3D p in ing
echnology) o c ea e bio eso bable an ioxidan ascula s en s [
15
]. The s en s p o ided an ioxidan
capabili ies ha a e bene icial o educe oxida i e s ess Ano he applica ion o an ioxidan compounds
o 3D p in ing applica ions was desc ibed by Lücking e al. [
16
]. In his wo k, ano he ype o 3D
p in ing, selec i e lase sin e ing, was used o p epa e ma e ials con aining an ioxidan o enhance cell
p oli e a ion [
16
]. They used UV-s able polyamide 12 ma e ial wi h an ioxidan p ope ies. Howe e ,
he na u e o he an ioxidan compound was no disclosed.
An in e es ing enewable and na u al compound wi h an ioxidan p ope ies is lignin (LIG). LIG
is a biopolyme p esen in he suppo issues o ascula plan s and p o ides chemical and mechanical
p o ec ion om ex e nal s esses [
17
]. Mo eo e , i has been epo ed ha LIG p esen s an ioxidan
and an imic obial p ope ies [
18
–
21
]. Among o he al e na i es, LIG-based ma e ials ha e a ac ed
he a en ion o esea che s [
17
,
22
–
27
]. Conside ing ha LIG is he second mos abundan polyme on
Ea h [
17
,
24
], he applica ions o LIG o de elop new ma e ials a e unexploi ed as only a 2% o he o al
LIG p oduc ion (ca. 70 million ons) is eused o special y p oduc s [
17
]. The es is used as bu ning
uel o ea ed as a was e [
17
,
28
]. Due o i s high a ailabili y and i s an imic obial and an ioxidan
p ope ies, LIG has po en ial o biomedical applica ions. These a e no he only ad an ages as LIG is
a ela i ely cheap o ob ain wi h p ices ca.
€
33 pe on [
29
]. To da e, he biomedical applica ions o his
biopolyme , emain ela i ely unexplo ed. A ew scien i ic a icles can be ound desc ibing i s use o
p epa e coa ings, nanopa icles o hyd ogels [21,30–32] o po en ial medical applica ions.
In he p esen wo k, we p opose he combina ion o LIG wi h PLA o c ea e ilamen ha can
be used o heal hca e FFF applica ions. These compounds we e combined ia ho mel ex usion.
The esul ing composi e ma e ials we e cha ac e ized and success ully used o used deposi ion
modeling applica ions. This app oach has po en ial o mul iple applica ions bu in he p esen s udy
we hypo hesized ha i can be used o wound d essing applica ions. The an ioxidan p ope ies o
he ma e ial could po en ially enhance wound healing. The esul ing polyme ic composi e ma e ial
has an ioxidan p ope ies and do no ely on he elease o an an ioxidan agen s. Acco dingly, he
ma e ial can p o ide localized and p olonged an ioxidan ac i i y. Finally, he p oposed me hod is
simple and allows combina ion o PLA wi h LIG and addi ional molecules such as an ibio ics. In his
way, he esul ing ma e ial can ha e ad anced p ope ies. Fo he p esen wo k we selec ed e acycline
(TC) as in his way he ma e ial can be used o p e en wound in ec ions.
2. Ma e ials and Me hods
2.1. Ma e ials
Ingeo PLA 3D850 biopolyme (PLA pelle s) was pu chased om Na u eWo ks LCC
(
Minne onka, MN, USA
). This PLA g ade (3D850) has been speci ically de eloped o FFF applica ions,
ha ing an excellen p ocessabili y and p in abili y.
LIG sample (BioPi a 100) was a so wood K a LIG acqui ed om UPM (Helsinki, Finland).
This LIG sample was used as p o ided (63–68% d y ma e con en ). Howe e , he highe mois u e
con en o his sample was conside ed o any ollowing calcula ions. An impo an pa o he
Pha maceu ics 2019,11, 165 3 o 14
cha ac e iza ion o his a oma ic polyme was kindly p o ided om he supplie . Klason LIG con en
(TAPPI T 222 om-02) was a ound 92% o he d y ma e and acid-soluble LIG (TAPPI UM 250) was
a ound 4% o he d y ma e . On he o he hand, he o al amoun o ca bohyd a es (SCAN-CM 71:09)
accoun ed o a ound 2% o he d y ma e and he con en o ino ganic pa icles (in e nal me hod,
700
◦
C) accoun ed o a ound 1% o he d y ma e . Mola mass o his LIG sample was 5000—6000 Da.
Finally, he pa icle size (median d
50
) was a ound 20
µ
m and i was analyzed using a Mas e size 3000
(Mal e n, UK).
Cas o oil used o a ach LIG and TC o he pelle s su ace was pu chased om Ransom Na u als
L d. (England, UK). TC was acqui ed om Honeywell Fluka
™
(Leices e shi e, UK). To pe o m
he elease s udy, Hyd oxyp opyl-me hyl cellulose (HPMC) was acqui ed by Colo con Limi ed
(Da o d, UK), asco bic acid (AA) was p o ided by DSM (Hee len, he Ne he lands), ween 80
(T80) was ob ained om Tokyo Chemical Indus y UK L d. (Ox o d, UK) and cu cumin (CUR), he
model d ug used in his s udy, was pu chased om Cayman Chemical (Michigan, USA). Finally,
poly( inyl alcohol) (PVA) ilamen was ob ained om Ul imake B.V., The Ne he lands (Diame e :
2.85 mm; Mel ing Tempe a u e: 163 ◦C).
S aphylococcus au eus (ATCC 6538; LGC S anda ds, Middlesex, UK) we e main ained on
c yop ese a i e beads in 10% glyce ol a
−
80
◦
C and cul i a ed in Muelle Hin on b o h (MHB)
a 37 ◦C when equi ed o he mic obiological assessmen s.
2.2. LIG-PLA Pelle s P oduc ion
The me hod used o p oduce hese LIG-PLA coa ed pelle s was p e iously desc ibed by Weisman
e al. [
10
]. B ie ly, 50 mL Falcon ube was illed wi h PLA pelle s (40 g). Then, cas o oil (40
µ
L)
was added in o he ube and i was o exed un il he pelle s we e p ope ly coa ed. Subsequen ly,
hese cas o oil co e ed pelle s we e placed in o a new 50 mL Falcon ube and he LIG o LIG and
e acycline powde we e added, and i was o exed again. Di e en ba ches con aining 0.5%, 1%,
2% and 3% (w/w) o LIG coa ing and 1% and 2% (w/w) o LIG and TC, espec i ely, we e p epa ed.
Addi ionally, cas o oil co e ed PLA pelle s and only PLA pelle s we e used as con ol samples o
make he ilamen s.
2.3. Ex usion o Filamen s
The Nex 1.0 ilamen ex ude (3de o, U ech , The Ne he lands) was used o p epa e he di e en
ilamen s. The empe a u e was adjus ed h ough a con ol panel posi ioned a he side o he ex ude
and i was be ween 170 and 190
◦
C, due o he exis ence o 4 hea e s. Mo eo e , he ex ude speed
was es ablished a 5.0 pm and he an speed was 70%. Addi ionally, 2.85 mm was selec ed as he
diame e o he ex uded ilamen s.
2.4. Fused Filamen Fab ica ion
Once he ilamen s we e ex uded, discs and squa es we e 3D-p in ed using an Ul imake 3
(Ul imake B.V., Gelde malsen, The Ne he lands) FFF sys em and Cu a
®
so wa e be ween 185 and
205
◦
C. The Ul imake 3 FFF sys em was equipped wi h wo ex ude s con aining a 0.4 mm nozzle. I is
impo an o no e ha his equipmen is a RepRap Open Sou ce FFF equipmen [
4
]. The mog a ime ic
analyses we e pe o med o asce ain he s abili y o LIG a he p ocess empe a u es (da a no shown),
showing ha a small amoun o LIG was deg aded du ing he p ocess (ca. 3%).
2.5. Ma e ial Cha ac e iza ion
2.5.1. Mic oscopy
The mo phologies o he ma e ials p epa ed using FFF as well as he ilamen s and pelle s we e
assessed using a Leica EZ4 D digi al mic oscope (Leica, We zla , Ge many).
Pha maceu ics 2019,11, 165 4 o 14
2.5.2. Con ac Angle Measu emen
The in luence o he LIG on he con ac angle o wa e wi h he su ace o he 3D p in ed ma e ials
(squa es) was assessed using an A ension The a equipmen (A ension The a, Biolin Scien i ic,
Go henbu g, Sweden). OneA ension so wa e analyzed esul s o gi e an indica ion o he we abili y
o he su ace. Measu emen s we e pe o med in iplica e.
2.5.3. The mal P ope ies
The glass ansi ion empe a u e (T
g
) o he 3D p in ed ma e ials as well as he pu e ma e ials
(PLA, LIG, e acycline and cas o oil) was measu ed using DSC Q100 di e en ial scanning calo ime e
(TA ins umen s, Bellingham, WA, USA). Scans we e un om 30 o 300
◦
C a 10
◦
C/min unde a
ni ogen low a e o 10 mL/min.
2.5.4. S abili y S udy
Fo he deg ada ion expe imen s, he 3D p in ed discs we e incuba ed a 37
◦
C in sc ew-capped
ials con aining 3 mL o phospha e-bu e ed saline (PBS) (pH = 7.4) o e a pe iod o 30 days.
The specimens incuba ed we e un in iplica e. A e di e en in e al imes, he discs we e sepa a ed
om he deg ada ion medium and he excess o i was emo ed wi h a issue pape and subsequen ly
hey we e d ied a 80
◦
C in an o en o 5 min. The deg ada ion medium o PBS was eplaced a e
each ime in e al and he mass loss was measu ed.
2.5.5. Mechanical P ope ies
The b eak s eng h o he di e en ilamen s ex uded we e e alua ed using a TA.XTplus ex u e
analyze (S able Mic o Sys ems, Su ey, UK) in comp ession mode. Pieces o ilamen s o 2 cm we e
cu and used o he es . Then, hese samples we e placed on wo aluminum blocks (wi h 1 cm o
sepa a ion) and a ape ed aluminum p obe (5.5 cm in leng h wi h a blun end o adius 1.0 mm) was
mo ed owa ds he pieces o ilamen s. The p obe mo ed a a speed o 2 mm
·
s
−1
wi h a maximum
dis ance o a el o 5 mm. The ilamen ailu e o ce was assumed o be he peak maximum o he
o ce-dis ance cu e.
2.6. PLA-LIG Mesh P oduc ion and CUR Release o Po en ial Wound Healing Applica ions
Di e en 3D p in ed meshes (0.4 mm hickness) made using he ilamen con aining 2% o LIG
we e used o CUR pe mea ion expe imen s. Figu e 1shows he di e en ypes o meshes ha we e
p epa ed and hei dimensions. Mo eo e , combined meshes we e p epa ed using a wo laye ed
dis ibu ion (0.4 mm each laye ). The i s laye con ained a PLA/LIG mesh and he second laye
was p in ed using PVA. Fo hese pu pose, ho izon al di usion cell sys em was used wi h some
modi ica ions. Each side o he sys em was employed as an indi idual eplica e o he di e en mesh
sys ems. Cu cumin was used as he model d ug o his es . Films we e p epa ed wi h HPMC and
CUR. Fo his pu pose, a solu ion con aining 10% (w/w) o HPMC and an excess o CUR was p epa ed
in e hanol-wa e (70% e hanol). The solu ion was cen i uged o emo e he non-dissol ed CUR.
Subsequen ly, ilms we e cas ed using his solu ion. Then, a 1 cm diame e co k-bo e was employed
o cu his ilm in o discs, which we e placed oge he he di e en 3D p in ed meshes in he cells.
Then, each cell o he sys em was illed wi h 3 mL o a solu ion con aining 10% (w/ ) o Tween 80
and 1 mg/mL o asco bic acid p epa ed in PBS, which was s i ed and he mos a ically main ained a
37
±
1
◦
C. Samples (
≤
0.5 mL) we e emo ed om he sampling a ms o he cells a p ede e mined
ime in e als and eplace wi h esh elease media. The concen a ion o CUR was e alua ed using a
UV- isible pla e eade (Powe Wa e XS Mic opla e Spec opho ome e , Bio-Tek, Winooski, VT, USA)
a a wa eleng h o 425 nm.
Pha maceu ics 2019,11, 165 5 o 14
Pha maceu ics 2019, 11, x FOR PEER REVIEW 5 o 14
con aining 10% (w/ ) o Tween 80 and 1 mg/mL o asco bic acid p epa ed in PBS, which was
s i ed and he mos a ically main ained a 37 ± 1 °C. Samples (≤0.5 mL) we e emo ed om he
sampling a ms o he cells a p ede e mined ime in e als and eplace wi h esh elease media.
The concen a ion o CUR was e alua ed using a UV- isible pla e eade (Powe Wa e XS
Mic opla e Spec opho ome e , Bio-Tek, Winooski, VT, USA) a a wa eleng h o 425 nm.
Figu e 1. Scheme o he di e en meshes p oduced using FFF.
2.7. An ioxidan Ac i i y
DPPH (2,2-diphenyl-1-pic ylhyd ozyl) adical was employed o measu e he an ioxidan
ac i i y o 3D p in ed ma e ials based on he adical sca enging p ope y o he LIG [33]. B ie ly,
3 mL o a DPPH solu ion dissol ed in me hanol (23.6 mg/L) was added o he 3D p in ed samples
(a squa e o 1 cm × 1 cm × 0.1 cm) placed in a 24-well pla e. A con ol sample o 23.6 mg/L o
DPPH in me hanol was also measu ed. The samples we e hen incuba ed in he da k o 300 min
a oom empe a u e. A p ede e mined ime in e als (each 60 min), 300 µL samples we e
collec ed and he well was immedia ely eplenished wi h an equi alen olume o me hanol. The
abso bance o he di e en solu ions was measu ed a 517 nm in iplica e using a UV– is pla e
eade (Powe Wa e XS Mic opla e Spec opho ome e , Bio-Tek, Winooski, VT, USA). The
esidual DPPH con en in he solu ion was calcula ed using Equa ion (1)
Residual DPPH con en (%) = 100 − 100 (A
0
− A
1
/A
0
) (1)
whe e A
0
is he abso bance o he con ol sample and A
1
is he abso bance in he p esence o he
sample a any ime. Dec eased abso bance o he eac ion indica es a s onge DPPH adical
sca enging ac i i y.
2.8. An imic obial P ope ies
The in i o mic obiological analysis was pe o med acco ding o he p e ious published
wo ks [34,35]. In b ie , a bac e ial suspension o S. au eus (1 × 10
8
c u mL
−1
) in PBS and
supplemen ed wi h 0.5% TSB (pH 7), was dilu ed (1:100) wi h PBS con aining 0.5% TSB. Replica e
samples o 3D p in ed squa es (1 cm × 1 cm × 0.1 cm) we e placed in indi idual wells o a 24-well
pla e and hen aliquo s o 1 mL o he dilu ed bac e ial suspension wi h a densi y o 1 × 10
6
c u
Figu e 1. Scheme o he di e en meshes p oduced using FFF.
2.7. An ioxidan Ac i i y
DPPH (2,2-diphenyl-1-pic ylhyd ozyl) adical was employed o measu e he an ioxidan ac i i y
o 3D p in ed ma e ials based on he adical sca enging p ope y o he LIG [
33
]. B ie ly, 3 mL o a
DPPH solu ion dissol ed in me hanol (23.6 mg/L) was added o he 3D p in ed samples (a squa e o
1 cm ×1 cm ×0.1 cm
) placed in a 24-well pla e. A con ol sample o 23.6 mg/L o DPPH in me hanol
was also measu ed. The samples we e hen incuba ed in he da k o 300 min a oom empe a u e.
A p ede e mined ime in e als (each 60 min), 300
µ
L samples we e collec ed and he well was
immedia ely eplenished wi h an equi alen olume o me hanol. The abso bance o he di e en
solu ions was measu ed a 517 nm in iplica e using a UV– is pla e eade (Powe Wa e XS Mic opla e
Spec opho ome e , Bio-Tek, Winooski, VT, USA). The esidual DPPH con en in he solu ion was
calcula ed using Equa ion (1)
Residual DPPH con en (%) = 100 −100 (A0−A1/A0) (1)
whe e A
0
is he abso bance o he con ol sample and A
1
is he abso bance in he p esence o he sample
a any ime. Dec eased abso bance o he eac ion indica es a s onge DPPH adical sca enging ac i i y.
2.8. An imic obial P ope ies
The
in i o
mic obiological analysis was pe o med acco ding o he p e ious published
wo ks [
34
,
35
]. In b ie , a bac e ial suspension o S. au eus (1
×
10
8
c u mL
−1
) in PBS and supplemen ed
wi h 0.5% TSB (pH 7), was dilu ed (1:100) wi h PBS con aining 0.5% TSB. Replica e samples o 3D
p in ed squa es (1 cm
×
1 cm
×
0.1 cm) we e placed in indi idual wells o a 24-well pla e and
hen aliquo s o 1 mL o he dilu ed bac e ial suspension wi h a densi y o 1
×
10
6
c u mL
−1
was
added comple ely co e ing he 3D p in ed squa es. The pla e was con inuously shaken in an o bi al
incuba o a 37
◦
C o 24 h. Then he samples we e emo ed om he 24-well pla e con aining he
bac e ial suspension and he non-adhe en bac e ia we e emo ed by se e al washing s eps, i s in PBS
(
1×10 mL
), and hen in qua e -s eng h Ringe ’s solu ion (QSRS) (3
×
10 mL) [
36
]. A e he wash
s ep, 3D p in ed squa es we e ans e ed in o esh QSRS (5 mL), sonica ed (15 min) and o exed
(30 s) o emo e adhe en bac e ia. The sonica ion echnique has p e iously been demons a ed no o

Pha maceu ics 2019,11, 165 6 o 14
a ec bac e ial iabili y o mo phology [37]. A iable coun o he QSRS was pe o med by he Miles
and Mis a se ial dilu ion echnique [
38
] ollowed by pla ing on o Muelle –Hin on aga o enume a e
he p e iously adhe ed bac e ia pe sample.
2.9. S a is ical Analysis
All da a we e exp essed as mean
±
s anda d de ia ion. Da a we e compa ed using a one-way
analysis o a iance (ANOVA), wi h Tukey’s HSD pos -hoc es . In all cases, p< 0.05 was he minimum
alue conside ed accep able o ejec ion o he null hypo hesis.
3. Resul s
3.1. PLA and LIG Composi e Ma e ial P epa a ion and Cha ac e iza ion
The an ioxidan capabili ies o LIG ha e been epo ed mul iple imes in he pas . Conside ing he
po en ial heal h bene i s associa ed wi h an ioxidan ma e ials, LIG has po en ial o be combined
wi h 3D p in able biocompa ible polyme s o biomedical applica ions. Fo his pu pose, PLA was
selec ed as he ideal ma e ial o be combined wi h LIG. I has been ex ensi ely used o FFF 3D p in ing
applica ions and i is biocompa ible and biodeg adable [7,9].
Ho mel ex usion was used o combine LIG and PLA o o m a composi e ma e ial. PLA was
supplied in pelle o m while LIG was supplied in powde o m. To ge a homogeneous mix u e o
bo h ypes o ma e ials, a coa ing app oach was used. Cas o oil was used o coa he PLA pelle s
and, subsequen ly, LIG powde was added o he mix u e. This me hod was desc ibed be o e o
combine PLA wi h chemo he apeu ic and an ibio ic d ugs. Figu e 2A shows he image o he LIG
coa ed pelle s con aining LIG concen a ions anging om 0.5% o 3% (w/w). When highe amoun s
o LIG we e added, he pelle s showed a highe coa ing deg ee. TC, an an ibio ic compound wi h
epo ed an ioxidan capabili ies, was combined wi h PLA and LIG. These pelle s con ained a 2%
(w/w) o TC and 1% (w/w) o LIG. The coa ing o hese pelle s was homogeneous, as i can be seen in
Figu e 2A. Finally, 3% was he maximum LIG loading ha was e alua ed, as, in his case, he pelle s
we e comple ely coa ed by LIG powde (Figu e 2A).
PLA was combined wi h LIG and TC success ully using ho mel ex usion. Figu e 2B shows
agmen s o he ob ained ilamen s. Mo eo e , he ilamen s we e success ully used o FFF applica ion,
as sshown in Figu e 2C. Squa es (1 cm
×
1 cm) we e success ully p in ed using he PLA/LIG
composi es. Howe e , he ilamen s could be used success ully o p epa e mo e complex geome ies
as i is illus a ed in Figu e 2D.
Pha maceu ics 2019, 11, x FOR PEER REVIEW 7 o 14
Figu e 2. Pho og aphs o : PLA and PLA coa ed pelle s (A); LIG and TC con aining PLA ilamen s
(B); LIG and TC con aining 1 cm × 1 cm squa es p epa ed using 3D p in ing (C); and di e en
shapes p in ed using he ilamen con aining 2% (w/w) LIG (D).
The p oposed me hod is a good al e na i e o combine PLA and LIG, as i p o ided a good
mix u e o bo h componen s and did no equi e he use o any sol en s. In he pas , LIG and PLA
ha e been combined using a cas ing me hod [39]. This me hod equi es he use o sol en s ha
could p esen oxici y issues o heal hca e applica ions.
LIG/PLA ilamen s showed lowe esis ance o ac u e han PLA ilamen s. Figu e 3A shows
ha he p esence o LIG in he ma e ial educed he maximum load ha he ma e ials can esis
be o e ac u e. The e we e no signi ican di e ences be ween he maximum load ob ained o
ma e ials con aining 0% and 0.5% (w/w) o LIG (p = 0.506). In e es ingly, he maximum load
d opped when he LIG con en was inc eased om 0.5% o 1% (w/w) (p < 0.05). Mo eo e , when
he LIG con en eached 3%, he esul ing ma e ials showed highe esis ance o ac u e (p < 0.05).
The su ace p ope ies o he 3D p in ed ma e ials we e e alua ed. Figu e 3B shows he
con ac angle o wa e wi h he ma e ial su ace. In e es ingly, he e we e no signi ican
di e ences be ween he con ac angles ob ained o PLA + cas o oil and he ma e ials con aining
up o 1% o LIG (p > 0.05). Cas o oil did no in luence in he con ac angle o he ma e ials. The
ob ained con ac angles o PLA and PLA/cas o oil showed no signi ican di e ences be ween
hem (p = 0.995). Howe e , he ma e ials showed a no iceable educ ion in hei we abili y when
he LIG con en was inc eased up o 2% (w/w). The measu ed con ac angle be ween ma e ials
con aining 2% and 3% o LIG we e signi ican ly lowe han he p e iously desc ibed ma e ials (p
< 0.05). Addi ionally, he ob ained con ac angles o hese ma e ials showed simila alues.
Figu e 3. Maximum load be o e ac u e o LIG con aining ilamen s (n = 5) (A); and con ac angle
o wa e wi h he su ace o 3D p in ed ma e ials ob ained using PLA/LIG composi es (n = 4) (B).
Figu e 2.
Pho og aphs o : PLA and PLA coa ed pelle s (
A
); LIG and TC con aining PLA ilamen s (
B
);
LIG and TC con aining 1 cm
×
1 cm squa es p epa ed using 3D p in ing (
C
); and di e en shapes
p in ed using he ilamen con aining 2% (w/w) LIG (D).
Pha maceu ics 2019,11, 165 7 o 14
The p oposed me hod is a good al e na i e o combine PLA and LIG, as i p o ided a good
mix u e o bo h componen s and did no equi e he use o any sol en s. In he pas , LIG and PLA
ha e been combined using a cas ing me hod [
39
]. This me hod equi es he use o sol en s ha could
p esen oxici y issues o heal hca e applica ions.
LIG/PLA ilamen s showed lowe esis ance o ac u e han PLA ilamen s. Figu e 3A shows
ha he p esence o LIG in he ma e ial educed he maximum load ha he ma e ials can esis be o e
ac u e. The e we e no signi ican di e ences be ween he maximum load ob ained o ma e ials
con aining 0% and 0.5% (w/w) o LIG (p= 0.506). In e es ingly, he maximum load d opped when he
LIG con en was inc eased om 0.5% o 1% (w/w) (p< 0.05). Mo eo e , when he LIG con en eached
3%, he esul ing ma e ials showed highe esis ance o ac u e (p< 0.05).
The su ace p ope ies o he 3D p in ed ma e ials we e e alua ed. Figu e 3B shows he con ac
angle o wa e wi h he ma e ial su ace. In e es ingly, he e we e no signi ican di e ences be ween
he con ac angles ob ained o PLA + cas o oil and he ma e ials con aining up o 1% o LIG (
p> 0.05
).
Cas o oil did no in luence in he con ac angle o he ma e ials. The ob ained con ac angles o PLA
and PLA/cas o oil showed no signi ican di e ences be ween hem (p= 0.995). Howe e , he ma e ials
showed a no iceable educ ion in hei we abili y when he LIG con en was inc eased up o 2% (w/w).
The measu ed con ac angle be ween ma e ials con aining 2% and 3% o LIG we e signi ican ly lowe
han he p e iously desc ibed ma e ials (p< 0.05). Addi ionally, he ob ained con ac angles o hese
ma e ials showed simila alues.
Pha maceu ics 2019, 11, x FOR PEER REVIEW 7 o 14
Figu e 2. Pho og aphs o : PLA and PLA coa ed pelle s (A); LIG and TC con aining PLA ilamen s
(B); LIG and TC con aining 1 cm × 1 cm squa es p epa ed using 3D p in ing (C); and di e en
shapes p in ed using he ilamen con aining 2% (w/w) LIG (D).
The p oposed me hod is a good al e na i e o combine PLA and LIG, as i p o ided a good
mix u e o bo h componen s and did no equi e he use o any sol en s. In he pas , LIG and PLA
ha e been combined using a cas ing me hod [39]. This me hod equi es he use o sol en s ha
could p esen oxici y issues o heal hca e applica ions.
LIG/PLA ilamen s showed lowe esis ance o ac u e han PLA ilamen s. Figu e 3A shows
ha he p esence o LIG in he ma e ial educed he maximum load ha he ma e ials can esis
be o e ac u e. The e we e no signi ican di e ences be ween he maximum load ob ained o
ma e ials con aining 0% and 0.5% (w/w) o LIG (p = 0.506). In e es ingly, he maximum load
d opped when he LIG con en was inc eased om 0.5% o 1% (w/w) (p < 0.05). Mo eo e , when
he LIG con en eached 3%, he esul ing ma e ials showed highe esis ance o ac u e (p < 0.05).
The su ace p ope ies o he 3D p in ed ma e ials we e e alua ed. Figu e 3B shows he
con ac angle o wa e wi h he ma e ial su ace. In e es ingly, he e we e no signi ican
di e ences be ween he con ac angles ob ained o PLA + cas o oil and he ma e ials con aining
up o 1% o LIG (p > 0.05). Cas o oil did no in luence in he con ac angle o he ma e ials. The
ob ained con ac angles o PLA and PLA/cas o oil showed no signi ican di e ences be ween
hem (p = 0.995). Howe e , he ma e ials showed a no iceable educ ion in hei we abili y when
he LIG con en was inc eased up o 2% (w/w). The measu ed con ac angle be ween ma e ials
con aining 2% and 3% o LIG we e signi ican ly lowe han he p e iously desc ibed ma e ials (p
< 0.05). Addi ionally, he ob ained con ac angles o hese ma e ials showed simila alues.
Figu e 3. Maximum load be o e ac u e o LIG con aining ilamen s (n = 5) (A); and con ac angle
o wa e wi h he su ace o 3D p in ed ma e ials ob ained using PLA/LIG composi es (n = 4) (B).
Figu e 3.
Maximum load be o e ac u e o LIG con aining ilamen s (n= 5) (
A
); and con ac angle o
wa e wi h he su ace o 3D p in ed ma e ials ob ained using PLA/LIG composi es (n= 4) (B).
The ma e ials we e analyzed using FTIR spec oscopy (da a no shown). No di e ences we e
obse ed in he spec a o pu e PLA and LIG con aining ma e ials. This is due o he lowe LIG loading
wi hin he ma e ials. DSC measu emen s we e pe o med o e alua e he in e ac ion be ween LIG and
PLA. Figu e 4A shows he DSC he mog ams o LIG, PLA, cas o oil and he 3D p in ed PLA/LIG
composi es. This igu e shows ha PLA/LIG composi es showed he same ansi ions ha can be
obse ed o PLA. The i s ansi ion obse ed was a glass ansi ion (T
g
) a ca. 65
◦
C. PLA mel ing
poin was obse ed a ca. 180
◦
C. When LIG was inco po a ed in o he ma e ial, a educ ion in he T
g
was obse ed (Figu e 4B,C). The ma e ials con aining 0.5% and 1% o LIG showed almos he same
T
g
alue as pu e PLA. Howe e , when LIG loading inc eased up o 2%, a T
g
educ ion was obse ed
(Figu e 4B,C). Composi e ma e ials con aining 3% o LIG showed simila beha io . In e es ingly, he
mel ing empe a u e o he ma e ials was no a ec ed by he LIG p esence. Finally, he s abili y s udies
showed ha he ma e ials did no lose weigh a e 30 days in PBS (p< 0.05) (da a no shown).
Pha maceu ics 2019,11, 165 8 o 14
Pha maceu ics 2019, 11, x FOR PEER REVIEW 8 o 14
The ma e ials we e analyzed using FTIR spec oscopy (da a no shown). No di e ences we e
obse ed in he spec a o pu e PLA and LIG con aining ma e ials. This is due o he lowe LIG
loading wi hin he ma e ials. DSC measu emen s we e pe o med o e alua e he in e ac ion
be ween LIG and PLA. Figu e 4A shows he DSC he mog ams o LIG, PLA, cas o oil and he 3D
p in ed PLA/LIG composi es. This igu e shows ha PLA/LIG composi es showed he same
ansi ions ha can be obse ed o PLA. The i s ansi ion obse ed was a glass ansi ion (T
g
)
a ca. 65 °C. PLA mel ing poin was obse ed a ca. 180 °C. When LIG was inco po a ed in o he
ma e ial, a educ ion in he T
g
was obse ed (Figu e 4B,C). The ma e ials con aining 0.5% and 1%
o LIG showed almos he same T
g
alue as pu e PLA. Howe e , when LIG loading inc eased up
o 2%, a T
g
educ ion was obse ed (Figu e 4B,C). Composi e ma e ials con aining 3% o LIG
showed simila beha io . In e es ingly, he mel ing empe a u e o he ma e ials was no a ec ed
by he LIG p esence. Finally, he s abili y s udies showed ha he ma e ials did no lose weigh
a e 30 days in PBS (p < 0.05) (da a no shown).
Figu e 4. DSC he mog ams ob ained o LIG, PLA, cas o oil and he esul ing PLA/LIG and TC
composi es (A); expanded iew o he he mog am be ween 60 and 76 °C (B); and T
g
a ia ion as
a unc ion o he LIG con en (C).
3.2. PLA and LIG Composi e An ioxidan and An imic obial P ope ies
Figu e 5A shows he adical sca enging ac i i y o PLA/LIG 3D p in ed composi es. The
p esence o LIG in he ma e ial ga e he ma e ial an ioxidan p ope ies. PLA and PLA + cas o
oil based ma e ials did no show a DPPH concen a ion educ ion o e ime. Mo eo e , ma e ials
con aining LIG showed highe adical sca enging ac i i y as he p esence o DPPH dec eased
o e ime. This indica es ha he p esence o LIG p o ides he an ioxidan ac i i y o he
ma e ials. As expec ed, ma e ials wi h highe LIG con en showed highe an ioxidan ac i i y as
hey we e mo e e icien in educing he DPPH concen a ion o e ime.
I has been epo ed p e iously ha LIG has an imic obial ac i i y. Acco dingly, he
an imic obial capabili ies o he 3D p in ed ma e ials we e e alua ed by s udying bac e ial
adhesion. The adhesion o S. au eus o he ma e ial was e alua ed (Figu e 5B). When compa ing
he bac e ial adhesion o PLA/cas o oil (blank) and he 3D p in ed ma e ial con aining 1% (w/w)
LIG, he e we e no signi ican di e ences (p = 0.980). Acco dingly, i can be es ablished ha he
p esence o 1% (w/w) LIG did no p o ide any an imic obial capabili ies o he ma e ial. Mo eo e ,
simila esul s we e ob ained o highe concen a ions o LIG (p > 0.05) (da a no shown).
Acco dingly, we can es ablish ha he selec ed concen a ions o LIG we e no adding any
an imic obial capabili ies o he composi e ma e ials. Howe e , he addi ion o he TC showed
signi ican educ ions in bac e ial adhe ence (Figu e 5B) (p = 0.001). TC is an an ibio ic compound
and acco dingly can educe he bac e ial load a ached o he su ace o he ma e ial.
Figu e 4.
DSC he mog ams ob ained o LIG, PLA, cas o oil and he esul ing PLA/LIG and TC
composi es (
A
); expanded iew o he he mog am be ween 60 and 76
◦
C (
B
); and T
g
a ia ion as a
unc ion o he LIG con en (C).
3.2. PLA and LIG Composi e An ioxidan and An imic obial P ope ies
Figu e 5A shows he adical sca enging ac i i y o PLA/LIG 3D p in ed composi es. The p esence
o LIG in he ma e ial ga e he ma e ial an ioxidan p ope ies. PLA and PLA + cas o oil based
ma e ials did no show a DPPH concen a ion educ ion o e ime. Mo eo e , ma e ials con aining LIG
showed highe adical sca enging ac i i y as he p esence o DPPH dec eased o e ime. This indica es
ha he p esence o LIG p o ides he an ioxidan ac i i y o he ma e ials. As expec ed, ma e ials wi h
highe LIG con en showed highe an ioxidan ac i i y as hey we e mo e e icien in educing he
DPPH concen a ion o e ime.
I has been epo ed p e iously ha LIG has an imic obial ac i i y. Acco dingly, he an imic obial
capabili ies o he 3D p in ed ma e ials we e e alua ed by s udying bac e ial adhesion. The adhesion
o S. au eus o he ma e ial was e alua ed (Figu e 5B). When compa ing he bac e ial adhesion o
PLA/cas o oil (blank) and he 3D p in ed ma e ial con aining 1% (w/w) LIG, he e we e no signi ican
di e ences (p= 0.980). Acco dingly, i can be es ablished ha he p esence o 1% (w/w) LIG did
no p o ide any an imic obial capabili ies o he ma e ial. Mo eo e , simila esul s we e ob ained
o highe concen a ions o LIG (p> 0.05) (da a no shown). Acco dingly, we can es ablish ha
he selec ed concen a ions o LIG we e no adding any an imic obial capabili ies o he composi e
ma e ials. Howe e , he addi ion o he TC showed signi ican educ ions in bac e ial adhe ence
(Figu e 5B) (p= 0.001). TC is an an ibio ic compound and acco dingly can educe he bac e ial load
a ached o he su ace o he ma e ial.
Pha maceu ics 2019, 11, x FOR PEER REVIEW 9 o 14
Figu e 5. Residual DPPH con en as a unc ion o ime o he LIG con aining composi es (n = 3)
(A); and bac e ial adhesion o he PLA/LIG and PLA/LIG/TC composi es (n = 3) (B).
3.3. 3D P in ed PLA and LIG Composi e Meshes o Po en ial Wound Healing Applica ions.
As desc ibed p e iously, an ioxidan ma e ials can be ex emely bene icial o heal hca e
applica ions such as wound healing. Acco dingly, he p oposed ma e ials can be easily in eg a ed
in wound d essings. Fo his pu pose, 3D p in ed meshes we e p epa ed using PLA/LIG
composi e ma e ials. These meshes can p o ide mechanical p o ec ion o he wound while
p o iding an ioxidan ac i i y. Due o hei design, soluble pa ches con aining d ugs can be
applied o he su ace o he mesh. The d ug can di use h ough he mesh po es o he wound.
An expe imen al se up was p epa ed o e alua e he deli e y o an an ioxidan and wound
healing model compound, CUR (Figu e 6A). Two di e en ypes o meshes we e p epa ed
con aining wo di e en g id sizes (1 and 1.5 mm) (Figu e 6B). As shown in Figu e 6B, he
ma e ials we e lexible and hus could adap o he su ace o he wound.
Figu e 6C shows he pe mea ion o CUR h ough he meshes. The 1.5 mm meshes p o ided
a slowe CUR elease han he con ol (CUR con aining ilm alone). This e ec was mo e
subs an ial when he mesh size was educed o 1 mm (Figu e 6D). Mo eo e , he elease a e could
be delayed by combining he mesh wi h a soluble PVA ilm. The PVA ilm could be p in ed in
combina ion wi h he LIG/PLA mesh using use deposi ion modeling equipmen equipped wi h
a dual ex ude . Figu e 6C shows how he inco po a ion o a PVA ilm wi h a LIG/PLA mesh
delayed CUR elease.
Figu e 6. Expe imen al se up used o measu e d ug di usion ough he 3D p in ed meshes (A);
pho og aphs o he 3D p in ed meshes made o PLA and 2% (w/w) LIG (B); and CUR elease
h ough 1.5 mm (C) and 1 mm (D) 3D p in ed meshes (n = 3).
Figu e 5.
Residual DPPH con en as a unc ion o ime o he LIG con aining composi es (n= 3) (
A
);
and bac e ial adhesion o he PLA/LIG and PLA/LIG/TC composi es (n= 3) (B).
Pha maceu ics 2019,11, 165 9 o 14
3.3. 3D P in ed PLA and LIG Composi e Meshes o Po en ial Wound Healing Applica ions
As desc ibed p e iously, an ioxidan ma e ials can be ex emely bene icial o heal hca e
applica ions such as wound healing. Acco dingly, he p oposed ma e ials can be easily in eg a ed
in wound d essings. Fo his pu pose, 3D p in ed meshes we e p epa ed using PLA/LIG composi e
ma e ials. These meshes can p o ide mechanical p o ec ion o he wound while p o iding an ioxidan
ac i i y. Due o hei design, soluble pa ches con aining d ugs can be applied o he su ace o he mesh.
The d ug can di use h ough he mesh po es o he wound. An expe imen al se up was p epa ed
o e alua e he deli e y o an an ioxidan and wound healing model compound, CUR (Figu e 6A).
Two di e en ypes o meshes we e p epa ed con aining wo di e en g id sizes (1 and 1.5 mm)
(Figu e 6B). As shown in Figu e 6B, he ma e ials we e lexible and hus could adap o he su ace o
he wound.
Figu e 6C shows he pe mea ion o CUR h ough he meshes. The 1.5 mm meshes p o ided a
slowe CUR elease han he con ol (CUR con aining ilm alone). This e ec was mo e subs an ial
when he mesh size was educed o 1 mm (Figu e 6D). Mo eo e , he elease a e could be delayed by
combining he mesh wi h a soluble PVA ilm. The PVA ilm could be p in ed in combina ion wi h he
LIG/PLA mesh using use deposi ion modeling equipmen equipped wi h a dual ex ude . Figu e 6C
shows how he inco po a ion o a PVA ilm wi h a LIG/PLA mesh delayed CUR elease.
Pha maceu ics 2019, 11, x FOR PEER REVIEW 9 o 14
Figu e 5. Residual DPPH con en as a unc ion o ime o he LIG con aining composi es (n = 3)
(A); and bac e ial adhesion o he PLA/LIG and PLA/LIG/TC composi es (n = 3) (B).
3.3. 3D P in ed PLA and LIG Composi e Meshes o Po en ial Wound Healing Applica ions.
As desc ibed p e iously, an ioxidan ma e ials can be ex emely bene icial o heal hca e
applica ions such as wound healing. Acco dingly, he p oposed ma e ials can be easily in eg a ed
in wound d essings. Fo his pu pose, 3D p in ed meshes we e p epa ed using PLA/LIG
composi e ma e ials. These meshes can p o ide mechanical p o ec ion o he wound while
p o iding an ioxidan ac i i y. Due o hei design, soluble pa ches con aining d ugs can be
applied o he su ace o he mesh. The d ug can di use h ough he mesh po es o he wound.
An expe imen al se up was p epa ed o e alua e he deli e y o an an ioxidan and wound
healing model compound, CUR (Figu e 6A). Two di e en ypes o meshes we e p epa ed
con aining wo di e en g id sizes (1 and 1.5 mm) (Figu e 6B). As shown in Figu e 6B, he
ma e ials we e lexible and hus could adap o he su ace o he wound.
Figu e 6C shows he pe mea ion o CUR h ough he meshes. The 1.5 mm meshes p o ided
a slowe CUR elease han he con ol (CUR con aining ilm alone). This e ec was mo e
subs an ial when he mesh size was educed o 1 mm (Figu e 6D). Mo eo e , he elease a e could
be delayed by combining he mesh wi h a soluble PVA ilm. The PVA ilm could be p in ed in
combina ion wi h he LIG/PLA mesh using use deposi ion modeling equipmen equipped wi h
a dual ex ude . Figu e 6C shows how he inco po a ion o a PVA ilm wi h a LIG/PLA mesh
delayed CUR elease.
Figu e 6. Expe imen al se up used o measu e d ug di usion ough he 3D p in ed meshes (A);
pho og aphs o he 3D p in ed meshes made o PLA and 2% (w/w) LIG (B); and CUR elease
h ough 1.5 mm (C) and 1 mm (D) 3D p in ed meshes (n = 3).
Figu e 6.
Expe imen al se up used o measu e d ug di usion ough he 3D p in ed meshes (
A
);
pho og aphs o he 3D p in ed meshes made o PLA and 2% (w/w) LIG (
B
); and CUR elease h ough
1.5 mm (C) and 1 mm (D) 3D p in ed meshes (n= 3).
4. Discussion
Human popula ion g ow h and indus ial de elopmen a e gene a ing an inc easing demand
o polyme ic ma e ials. The majo i y o hese ypes o ma e ials a e de i ed om he pe ochemical
indus y and, acco dingly, hey ha e an eno mous impac on he en i onmen [
40
]. The e o e,
he scien i ic communi y is de eloping g een and sus ainable al e na i es o he adi ional polyme ic
ma e ials [
17
]. As men ioned p e iously, LIG has po en ial o be used as a unc ional addi i e due o
i s in e es ing p ope ies.
The an ioxidan p ope ies o LIG can be used o po en ial heal hca e ela ed applica ions such as
wound healing. Reac i e oxygen species a e s ongly linked o he pa hogenesis o ch onic wounds [
41
].
Acco dingly, as an ioxidan ma e ials con ibu e o educe he concen a ion o hese species, hey ha e
po en ial o be applied as wound healing ma e ials. Mo eo e , i has been shown ha LIG nano ib ous
d essings con ibu e o wound healing [42].