Kac inskáe al.
Jou nal o Nanobio echnology (2023) 21:80
h ps://doi.o g/10.1186/s12951-023-01822-5
RESEARCH
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Open Access
Jou nal o Nanobio echnology
Accelula nano ib ous bilaye sca old
in apene a ed wi hpolydopamine ne wo k
andimplemen ed in oa ull- hickness wound
o awhi e-pig model a ec s in lamma ion
andhealing p ocess
Ka a ína Kac inská1, Ve onika Pa liňáko á1, Pe Poláček1, Lenka Michlo ská1, Ve onika He ka Blahno á2,
E a Filo á2, Ma in Knoz3,4, Bře isla Lipo ý1,3, Jakub Holoubek3, Ma in Faldyna5, Zdeněk Pa lo ský6,
Monika Víceno á5, Michaela C ano á7, Jiří Ja ko ský7 and Lucy Voj o á1*
Abs ac
T ea men o comple e loss o skin hickness equi es expensi e cellula ma e ials and limi ed skin g a s used as
empo a y co e age. This pape p esen s an acellula bilaye sca old modi ied wi h polydopamine (PDA), which is
designed o mimic a missing de mis and a basemen memb ane (BM). The al e na e de mis is made om eeze-
d ied collagen and chi osan (Coll/Chi ) o collagen and a calcium sal o oxidized cellulose (Coll/CaOC). Al e na e BM
is made om elec ospun gela in (Gel), polycap olac one (PCL), and CaOC. Mo phological and mechanical analyzes
ha e shown ha PDA signi ican ly imp o ed he elas ici y and s eng h o collagen mic o ib ils, which a o ably
a ec ed swelling capaci y and po osi y. PDA signi ican ly suppo ed and main ained me abolic ac i i y, p oli e a ion,
and iabili y o he mu ine ib oblas cell lines. The in i o expe imen ca ied ou in a domes ic La ge whi e pig model
esul ed in he exp ession o p o-in lamma o y cy okines in he i s 1–2 weeks, gi ing he idea ha PDA and/o CaOC
igge he ea ly s ages o in lamma ion. O he wise, in la e s ages, PDA caused a educ ion in in lamma ion wi h he
exp ession o he an i-in lamma o y molecule IL10 and he ans o ming g ow h ac o β (TGFβ1), which could sup-
po he o ma ion o ib oblas s. Simila i ies in ea men wi h na i e po cine skin sugges ed ha he bilaye can be
used as an implan o ull- hickness skin wounds and hus elimina e he use o skin g a s.
Keywo ds Bilaye , Chi osan, Collagen, Oxidized cellulose, Polydopamine, Wound healing
*Co espondence:
Lucy Voj o á
lucy. oj [email p o ec ed].cz
1 CEITEC – Cen al Eu opean Ins i u e o Technology, B no Uni e si y
o Technology, Pu kyňo a 656/123, 612 00 B no, Czech Republic
2 Ins i u e o Expe imen al Medicine o he Czech Academy o Sciences,
Vídeňská142 20, 1083 P ague 4, Czech Republic
3 Depa men o Bu ns and Plas ic Su ge y, Facul y o Medicine, Ins i u ion
Sha ed Wi h Uni e si y Hospi al B no, Masa yk Uni e si y, Jihla ská, 20,
625 00 B no, Czech Republic
4 Depa men o Plas ic and Aes he ic Su ge y, Facul y o Medicine, S .
Anne’s Uni e si y Hospi al, Masa yk Uni e si y, Pekařská, 664/53, 602
00 B no, Czech Republic
5 Ve e ina y Resea ch Ins i u e, Hudco a 296/70, 621 00 B no, Czech
Republic
6 Ins i u e o Pa hology, Facul y o Medicine, Uni e si y Hospi al B no,
Masa yk Uni e si y, B no 625 00, Czech Republic
7 Ins i u e o Bios a is ics and Analyses, Facul y o Medicine, Masa yk
Uni e si y, Kamenice 5, 625 00 B no, Czech Republic
Page 2 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
Backg ound
Wound healing is an in eg a ed and complex p ocess ha
begins immedia ely a e inju y and in ol es he elease
o a la ge numbe o egula o y molecules, including
p o-in lamma o y cy okines, g ow h ac o s, and low
molecula weigh compounds om he se um o inju ed
blood essels and deg anula ed pla ele s [1, 2]. The epi-
de mal de ec i sel is called a supe icial wound, a de ec
in heepide mis and de mis oge he wi h damage o
blood essels, swea glands, e c. is called a pa ial hick-
ness wound, while damage o he subcu aneous a laye
is called a ull hickness wound and i leads o ex en-
si e loss o skin, hai ollicles, and glands [3]. The e a e
many s udies ha a e applied wi h ex ensi e esea ch o
he ea men o pa ial and ull- hickness wounds using
di e en ma e ials made o po ous oams, hyd ogels, o
nano ib ous laye s o syn he icma e ials (poly(e hylene
glycol) (PEG), poly(ε-cap olac one) (PCL), poly(lac ic-co-
glycolic acid) (PLGA), poly(lac ic acid) (PLA), poly( inyl
alcohol) (PVA), polyu e hane ilms, o silk ib oin) [4–6]
and/o na u al ma e ials based on collagen (Coll), gela in
(Gel), cellulose, algina e, chi osan, hyalu onan, ib in o
ucoidan ma e ials [7–9].
Coll has been widely used in many applica ions
because he na u ally occu ing p o ein consis s o h ee
α-domains (polypep ide chains) ha p o ide he main
mechanical suppo o cell a achmen and has excellen
biocompa ibili y and biodeg adabili y[10–14]. Howe e ,
Coll-based sca olds ace apid biodeg ada ion a es and
low mechanical s eng h. Chemical c oss-linking is he
mos e ec i e s a egy o p omo e s abili y. Ca bodiim-
ides ha e been widely in es iga ed as sui able c osslink-
e s o collagen sca olds [15–17]. Ano he possibili y is
he combina ion o Coll wi h na u al and/o syn he ic
ma e ials, which b ings new unc ional possibili ies o
issue enginee ing applica ions [18]. Chi osan (Chi ) is
a biodeg adable, non- oxic, and an ibac e ial ma e ial
wi h ahomeos a ic e ec . Chi is o en used in combi-
na ion wi h Coll; i accele a es ib oblas o ma ion and
enhances ea ly phase eac ions ela ed o healing [19,
20]. In ou p e ious s udies, [21, 22] we e alua ed Coll/
Chi sca olds en iched wi h ib oblas g ow h ac o 2
(FGF2) and u he in combina ion wi h selenium nano-
pa icles (SeNPs). The esul s showed suppo o ib o-
blas a achmen and me abolic ac i i y. In addi ion, he
sca olds exhibi ed an ibac e ial ac i i y agains h ee
s ains o bac e ia, Esche ichia Coli (E. coli), S aphylo-
coccus au eus (S. au eus), and me hicillin- esis an S.
au eus (MRSA). Chi can be p ocessed in a ious o ms
such as ilms, hyd ogels, ibe s, powde s, and mic o/
nanopa icles used in skin issue enginee ing [23, 24].
Oxidized cellulose is a biodeg adable polyme , wi h
non-immunogenici y, and i p omo es he healing o
ch onic wounds [25, 26]. In combina ion wi h Coll, i
educes p o-in lamma o y in e leukins, eac i e oxygen
species, and binds o me al ions wi h inc easing concen-
a ions o g ow h ac o s and p o einase inhibi o s [27].
The addi ion o calcium sal o oxidized cellulose (CaOC)
o elec ospun nano ibe s p o ided a unique inhibi-
o y e ec on E. coli bac e ia [28]. Poly(ε-cap olac one)
(PCL) is a syn he ic, biocompa ible, linea alipha ic poly-
es e ha is hyd ophobic, i deg ades ela i ely slowly,
and has good mechanical p ope ies. PCL sca olds ha e
been used as in i o issue implan s o a ious medical
applica ions and ha e shown g ea po en ial o wound
healing, bone issue enginee ing, ca dio ascula issue
enginee ing, and ne e egene a ion [29, 30]. Dopamine
is a molecule ha o ms na u al adhesion be ween he
ma e ial su aces o s icks small molecules. I is syn he-
sized in he body by cells and has an amino acid sequence
simila o ha o mussel p o ein, which has he abili y o
bind o many su aces in an aqueous en i onmen [31].
An a ac i e p ope y o dopamine is i s au o-polyme -
iza ion, which has been epo ed o occu in T is bu e
wi h pH o 8.5, whe e dopamine leads o polydopamine
(PDA) ilms and nano ibe s [32–34].
In ecen yea s, s udies based on mul ilaye sca olds in
he ea men o ull- hickness wounds ha e ad an ages
o e a single laye d essing because hey can unc ionally
eplace bo h de mal and epide mal componen s. Acellu-
la bilaye ma e ials we e p epa ed by a combina ion o
na u al and syn he ic ma e ials, e.g., Chi /PCL nano i-
b ous ma s, PLLA-mic opo ous disc [35]. Fu he mo e,
he Coll/Chi sca old en iched wi h ecombinan human
ascula endo helial g ow h ac o ( hVEGF) and an i-
bac e ial gen amicin we e encapsula ed in PLGA mic o-
sphe es [36]. A ilaye Chi -based sca old was p epa ed
o mo e accu a ely eplica e ull- hickness skin s ia ion
han a single o bilaye sca old, which equi ed weeks
o co-cul u e o ib oblas s and ke a inocy es o achie e
simila s ia ion[37]. The e a e many o he exis ing s ud-
ies ha conside he po en ial use o acellula mul ilay-
e ed sca olds, no only in skin issue, [38–40] bu also in
ascula issue [41] and bone issue enginee ing [42].
This s udy aims o de elop an acellula PDA-mod-
i ied bilaye sca old and o enhance mechanical
and biological suppo in ull- hickness po cine skin
wound econs uc ion. The bilaye is made o po ous
Coll/polysaccha ide oam (Chi o CaOC) wi h he aim
o mimicking a de mis-like s uc u e and a basal mem-
b ane-like s uc u e, cha ac e ized by a nano ib ous
laye made o biocompa ible polyme s gela in, PCL,
and CaOC. The PDA-modi ied bilaye signi ican ly
changes mechanical p ope ies and p omo es s abili y,
Page 3 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
leading o di e en wa e abso p ion and ma e ial
mo phology. These changes also allowed cells o p o-
li e a e wi h main ained iabili y. In i o e alua ion
o cy o oxici y and me abolic and p oli e a ion ac i -
i y o mu ine ib oblas s demons a ed a non-cy o oxic
e ec o implan ed bilaye s. In his s udy, he healing
p ocess was moni o ed o a longe pe iod o ime, as
well as he e ec o PDA a e ansplan a ion, which
shows he his ological analysis o in lamma o y and
an i-in lamma o y cy okines. PDA can enhance he
in lamma o y phase a he beginning o wound heal-
ing and shows possible suppo o he expansion o
g ow h ac o and an i-in lamma o y cy okines in he
middle and la e s ages o wound healing compa ed o
na i e po cine skin ea men .
Me hods
Ma e ials andchemicals
Bo ine Collagen ype I, 8w .% aqueous solu ion (Coll,
Collado s. .o., B no, Czech Republic), chi osan om
sh imp shells, 70% DDA, low iscosi y (Chi , Sigma-
Ald ich, Da ms ad , Ge many), calcium sal o oxidized
cellulose–deg ee o oxida ion 16–24% and Mn = 350kg/
mol (CaOC, Syn hesia, Pa dubice, Czech Republic),
ace ic acid (99%, Pen a s. .o, Ch udim, Czech Republic),
poly(ε-cap olac one) (PCL, 80 kg/mol), gela in (Gel,
Type B, Bio eagen , powde om bo ine skin), N-(3-
Dime hylaminop opyl)-N´-e hylca bodiimide hyd o-
chlo ide (EDC), N-hyd oxysuccinimide (NHS), 98%
dopamine hyd ochlo ide, is (hyd oxyme hyl) ami-
nome hane hyd ochlo ide, e hanol p.a. 99.8%, sodium
phospha e dibasic o molecula biology (≥ 98,5%),
sodium chlo ide, calcium chlo ide, sodium phospha e
dibasic dodecahyd a e (Na2HPO4 ·12H2O), po assium
dihyd ogen phospha e (KH2PO4), po assium chlo-
ide (KCl), collagenase om Clos idium his oly i-
cum, lysozyme human, he mu ine ib oblas cell lines
3T3-A31, Dulbecco’s modi ied eagle medium DMEM
(D6429), e al bo ine se um FBS (F7524), 2′,7′-bis
(2-ca boxye hyl)-5(6)-ca boxy luo escein ace oxym-
e hyl es e (BCECF-AM), p opidium iodide (P4864),
(all om Sigma Ald ich, Da ms ad , Ge many), penicil-
lin/s ep omycin (15140–122) and DiOC6(3) (D273),
(Li e Technologies, Eugene, OR, USA), oc enidine solu-
ion (Oc enisep ®, Schülke, Ge many), Bu omido ®
inj. (bu o phanol a a e, Vé oquinol, Czech epub-
lic), Domi o ®, Mede omidine, O ion co po a ion,
Finland) P opo ol® (P opo olum 1%, F esenius Kabi
Deu schland, Bad Hombu g, Ge many), Me acam®
(meloxicam, Boeh inge Ingelheim Ve medica, Ingel-
heim/Rhein, Ge many), En oxil® (En o loxacin, K ka,
No o mes o, Slo enia) Be adine®, (2.5% solu ion o
po idone iodine, EGIS Pha maceu icals PLC, Buda-
pes , Hunga y) we e used as ecei ed wi hou u he
pu i ica ion.
P epa a ion o samples
P epa a ion o po ous oams andc oss‑linked bilaye s
Po ous oams we e p epa ed acco ding o p e ious wo k
[43]. B ie ly, he calcula ed amoun o Coll (0.5 w .%)
and sui able polysaccha ide (0.5 w .%) in he weigh
a io o 1:1 was slowly homogenized. Ma e ial suspen-
sions we e eeze-d ied on an Epsilon 2-10D machine
(Ma in Ch is , Os e ode am Ha z, Ge many). A ib ous
laye was elec ospun on he su ace o he lyophilized
po ous oam acco ding o [28] modi ied wi h he addi-
ion o PCL. Nano ibe s we e p epa ed as ollows: he
Gel/PCL/CaOC 70/30/10 polyme solu ion was p epa ed
in concen a ed glacial ace ic acid and s i ed o e nigh .
Elec ospinning was pe o med using a labo a o y Nano-
spide NS LAB 500 machine (Depa men o Physical
Elec onics, Masa yk Uni e si y, B no, Czech Repub-
lic). The se ing pa ame e s we e as ollows: low a e
o 25mm‧min−1, applied ol age o 60kV, he dis ance
be ween he spinning and collec ing elec ode was se o
15cm, and he spinning elec ode was o a ed a a speed
o 5 pm. The ambien condi ions we e 23°C, 980kPa,
and 40% humidi y. A c oss-linking agen o he ca bodi-
imide sys em in e hanol (EDC/NHS in a mola a io 2/1)
was used o c oss-link he po ous oam and he nano i-
b ous laye . A e 2h o he c oss-linking p ocess, he
bilaye was washed wice wi h 0.1M Na2HPO4 ollowed
h ee imes wi h ul apu e wa e o emo e by-p oduc s.
Subsequen ly, he bilaye s we e eeze-d ied and s o ed
in desicca o s p io use.
P epa a ion o PDA‑coa ed c oss‑linked bilaye s
Po ous oams and nano ib ous laye s we e p epa ed
as desc ibed in 3.2.1. B ie ly, a nano ib ous laye was
elec ospun on he eeze-d ied po ous oam and bo h
pa s we e c oss-linked and washed a e 2h. A solu-
ion o dopamine hyd ochlo ide (2mg.mL−1 in 0.01M
T is HCl) was p epa ed be o e he second eeze-d y-
ing p ocess. T is HCl was used as an ini ia o o dopa-
mine polyme iza ion, esul ing in black polydopamine
(PDA) [31]. C oss-linked bilaye s we e imme sed in a
solu ion o  dopamine hyd ochlo ide and main ained
o ano he 24h unde ae obic condi ions. The PDA-
in apene a ed samples we e hen washed 5 imes in
wa e o emo e esidual unbound dopamine, and sub-
sequen ly he samples we e lyophilized again. Samples
we e always p epa ed ei he in a olume o 500μL in
24-well pla es o in i o es ing (only po ous oams),
o in a olume o 80mL in 12 × 12cm squa e plas ic
Page 4 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
pla es o biomechanical e alua ion (bilaye s). All
ypes o p epa ed samples a e summa ized in Table1.
An explana ion o sample abb e ia ions is as ollows:
The le e N indica es he p esence o a c oss-linked
nano ib ous laye (e.g., he abb e ia ion o Coll/Chi -
N/PDA belongs o he bilaye o med by Coll/Chi
oam wi h c oss-linked nano ibe s, coa ed wi h PDA).
The le e s NX indica e he p esence o a non-c oss-
linked nano ib ous laye , as i can be seen in Table1.
Sample cha ac e iza ion
S uc u e andmo phology
A scanning elec on mic oscope MIRA3 (TESCAN,
B no, Czech Republic) was used o s udy he mo -
phology and adhesion o he p epa ed bilaye s. Images
we e aken in a seconda y elec on emission mode,
he scan mode was DEPTH, he beam densi y was
10 and hehigh ol age was 10kV. The wo king dis-
ance was se o 15mm. The su ace o he samples
was coa ed wi h a 20nm hin laye o Au/Pd using EM
ACE 600 (Leica Mic osys ems, We zla , Ge many).
The po e size was cha ac e ized om SEM images
using ImageJ so wa e and SEM Image Po e Ex ac o
(SEMIPE). A minimum o i e and a maximum o en
images wi h he same esolu ion we e aken om each
sample. F om each image, 40–90 po es we e meas-
u ed. Da a we e e alua ed using a 2-sample T- es ,
which assumes unequal a iances and unequal sam-
ple sizes. The le el o signi icance was se a *p < 0.05
**p < 0.01and ***p < 0.001.
Fou ie ‑ ans o med in a ed s udy esul s
A Fou ie - ans o med in a ed spec oscopy (FTIR)
wi h a enua ed o al e lec ance (ATR-FTIR, Ve ex
70/70 , B uke , Bille ica, MA, USA) was pe o med o
cha ac e ize ma e ial composi ion o po ous oam wi h
and wi hou PDA and ma e ial composi ion o he PDA
bilaye s. P esen ed ATR-FTIR spec a we e aken om
a e aging 32 scans wi h a spec al esolu ion o 2 cm−1.
The displayed spec a in he wa enumbe ange o 4000–
500 cm−1we e no malized using min–max no maliza ion
(OPUS so wa e, B uke , Bille ica, MA, USA). The ATR-
FTIR spec a we e measu ed unde e acua ed condi ions
om all samples, each placed on a diamond ATR c ys al.
Dynamic mechanical analysis
An RSA G2 dynamic mechanical analyze (TA Ins u-
men s Inc., New Cas le, USA) was used o measu e he
ensile p ope ies o p epa ed bilaye s. The p epa ed
Table 1 Summa y o p epa ed samples
* All non-c oss-linked bilaye s a e excluded om ollowing expe imen s due o he low mechanical p ope ies and a emen ioned only in SEM obse a ion
Po ous oam composi ion Abb e ia ion o
oam
Collagen oam Coll
Collagen/Chi osan oam Coll/Chi
Collagen/CaOC oam Coll/CaOC
Po ous oam coa ed wi h PDA Abb e ia ion o
oam
Collagen oam coa ed wi h PDA Coll/PDA
Collagen/Chi osan coa ed wi h PDA Coll/Chi /PDA
Collagen/CaOC coa ed wi h PDA Coll/CaOC/PDA
Non-c oss-linked bilaye s* Abb e ia ion o
bilaye
Collagen bilaye ( oam + nano ibe s) Coll-NX
Collagen/Chi osan bilaye ( oam + nano ibe s) Coll/Chi -NX
Collagen/CaOC bilaye ( oam + nano ibe s) Coll/CaOC-NX
C oss-linked bilaye s Abb e ia ion o
bilaye
Collagen bilaye ( oam + nano ibe s) Coll-N
Collagen/Chi osan bilaye ( oam + nano ibe s) Coll/Chi -N
Collagen/CaOC bilaye ( oam + nano ibe s) Coll/CaOC-N
C oss-linked bilaye s coa ed wi h PDA Abb e ia ion o
bilaye
Collagen bilaye ( oam + nano ibe s and PDA coa ing) Coll-N/PDA
Collagen/Chi osan bilaye ( oam + nano ibe s and PDA coa ing) Coll/Chi -N/PDA
Collagen/CaOC bilaye ( oam + nano ibe s and PDA coa ing) Coll/CaOC-N/PDA
Page 5 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
samples we e cu in o s ips wi h a leng h o 40mm and a
wid h o 10mm. Thickness a ies wi h he ype o sample
(0.20mm–0.60mm) and samples we e measu ed wi h
Digi al Calipe 0–150mm (G oningen, Ne he lands). The
i s biomechanical es s we e pe o med a oom em-
pe a u e a 23°C and he second es in ol ed cons an
hyd a ion condi ions wi h phospha e bu e a 36°C in
abuil chambe su ounding he g ip. Be o e each meas-
u emen , 10min o swelling was allowed o each sam-
ple o swell he bilaye s. The ela ionship be ween s ess
and s ain is shown along wi h he co esponding elas ic
modulus. Da a analysis using Mic oso Excel was used
o he s a is ical e alua ion o i e samples o he same
bilaye . Da a we e e alua ed using a 2-sample T- es ,
which assumes unequal a iances and unequal sample
sizes. The le el o signi icance was se a **p < 0.01and
***p < 0.001.
Swelling capaci y
The bilaye s we e cu in o 1 × 2cm s ips and imme sed
in a wa e solu ion o es hei hyd oly ic s abili y unde
ambien condi ions. Each sample was weigh ed be o e
imme sion (Wi). The weigh o swollen samples (Ws) was
also eco ded a e gen ly emo ing he su ace wa e
wi h il e pape a se e al in e als: 1, 2, 5, 10, 15, 20, 30,
45, 60, 90, 120, 150, and 180min. A swelling a io was
calcula ed o de ine he exac amoun o swelling caused
by wa e abso p ion, and he swelling cu e was ob ained.
The swelling a io was calcula ed acco ding o Eq(1)
The samples we e measu ed in iplica es and he
esul s a e shown as mean ± s anda d de ia ion.
Enzyma ic s abili y
Collagenase om Clos idium his oly icum was used
o in es iga e in i o deg ada ion s udies o chemically
c oss-linked bilaye s. Deg ada ion was ca ied ou in p e-
pa ed phospha e bu e ed saline (PBS) a physiological pH
o 7.4 a 37°C. A e one hou o swelling, samples we e
emo ed om he PBS, subsequen ly weigh ed, and placed
in he collagenase solu ion (c = 2.2mg∙L−1). A e e e y 2,
4, 8, 24, 48, 72, 96, 120 and 144h, excess PBS was blo ed
on o he il e pape , ollowing he weigh no a ion, as well
as he pe cen age o weigh loss calcula ion using Eq.(2)
(1)
Swelling Ra io
=
W
s
Wi
(2)
Weigh Loss
=100 −

Wi·100
Ws
[%
]
whe e Ws ep esen s he weigh o he sca old a e 1h
o swelling and Wi ep esen s he weigh o he diges ed
sca old. Th ee measu emen s o each ype o sample
we e eco ded and shown as mean ± s anda d de ia ion.
In i o cy o oxici y assessmen
Mu ine ib oblas cell lines 3T3-A31 we e cul u ed
in cul u e medium con aining DMEM (high glucose,
D6429, Sigma-Ald ich, S . Louis, MO, USA), 10% FBS,
and 1% penicillin/s ep omycin. 70,000 cells/sca old
(wi h a diame e o 10mm and a heigh o 4–5mm) we e
seeded o ape iod o 14days.
Me abolic ac i i y was de e mined by he CellTi e
96® Aqueous One Solu ion Cell P oli e a ion (MTS)
me abolic assay (CellTi e 96® Aqueous One Solu-
ion Cell P oli e a ion Assay, P omega co p., Madison,
WI, USA), whe e he MTS e azolium compound was
added di ec ly o he cell cul u e medium in a 1:5 a io.
Me abolically ac i e cells educed he MTS eagen and
gene a ed acolo ed o mazan dye ha is soluble in cell
cul u e medium. Fo mazan dye was quan i ied by meas-
u ing he abso bance a 490nm, e e ence 690nm using
Tecan In ini e M200 P o. The samples we e ca ied ou
in biological quad uplica es; he esul s a e shown as
mean ± s anda d de ia ion.
The Quan -iT™ dsDNA Assay Reagen (In i ogen)
assay de e mined cell p oli e a ion, as i quan i ied he
amoun o double-s anded DNA. The assay con ains
a luo escen dye ac i a ed once i is bound o dsDNA.
Fluo escence was measu ed a λex = 485 nm and
λem = 523nm. The samples we e ca ied ou in biological
quad uplica es; he esul s a e shown as mean ± s anda d
de ia ion.
Cell iabili y was assessed by li e-dead s aining o h ee
samples. 2′,7′-bis (2-ca boxye hyl)-5(6)-ca boxy luo es-
cein ace oxyme hyl es e (BCECF, Sigma-Ald ich, Sain
Luis, MO, USA) was used o isualize he memb anes o
li ing cells and p opidium iodide o isualize he nuclei o
dead cells. Samples we e obse ed using a Zeiss LSM 880
Ai yscan con ocal mic oscope. Exci a ion/emission was
se as ollows: BCECF λex = 488nm/λem = 505–545nm,
PI λex = 560nm/λem ˃ 575nm.
The cell dis ibu ion on he sca old and he mo phol-
ogy we e obse ed using 3,3’-Dihexyloxaca bocyanine
Iodide (In i ogen™) DiOC6(3)/p opidium iodide (The -
moFishe Scien i ic™) s aining. Exci a ion/emission
was se as ollows: DiOC6(3) λex = 488nm/λem = 505–
545nm, PI λex = 560nm/λem ˃ 575nm. The signal om
he nuclei was u he used o de e mine he dep h o
pene a ion o he cell in o he sca old.

Page 6 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
The s a is ical signi icance was pe o med using one-
way analysis o a iance (ANOVA) in Sigma S a so wa e
3.5 (Sys a So wa e, Cali o nia, USA).
In i o expe imen
The in i o expe imen al pa was pe o med in a o al
o 2 emale pigs (Sus Sc o a domes icus) o he p oduc-
ion hyb id line wi h an ini ial weigh o 70 ± 5kg. All
phases o he expe imen las ed 6 mon hs. The pigs
we e supplied by a local p oduc ion company app o ed
by he Minis y o Ag icul u e o he Czech Republic
and housed a he Ve e ina y Resea ch Ins i u e (B no,
Czech Republic) in expe imen al s ables ce i ied by
he Minis y o Ag icul u e o he Czech Republic. The
s udy was ca ied ou acco ding o he Decla a ion o
Helsinki and was app o ed by he Ins i u ional Re iew
Boa d o he Ve e ina y Resea ch Ins i u e (p o ocol code
12/2016 wi h app o al om 21 Ap il 2016) and by he
B anch Commission o Animal Wel a e o he Minis y
o Ag icul u e o he Czech Republic (pe mission num-
be 34715/2016-MZE-17214 om 15 June 2016). Upon
a i al a he esea ch acili y, he pigs we e housed o
wo weeks p io o he expe imen al p ocedu e challenge
and housed indi idually in s ainless-s eel cages loca ed in
isola ed ooms wi h con olled egime and independen
en ila ion. Rooms we e kep a a empe a u e o 21°C,
a ela i e humidi y in he ange o 40–60%, and a en ila-
ion o app oxima ely 15 ai changes pe hou .
All su gical p ocedu es we e pe o med unde gen-
e al anes hesia. P e-medica ion and analgesia du ing he
su ge y we e pe o med by Bu omido inj. (bu o phanol
a a e) a a dose o 0.1mg‧kg−1 b.w. s.c. Anes hesia was
pe o med wi h Mede omidine a a dose o 0.5mg‧kg−1
body weigh . Fu he mo e, gene al anes hesia was main-
ained h oughou he su ge y by con inuous admin-
is a ion o P opo ol 1% a a dose (8–15mg‧kg−1 b.w.
i. .). Immedia ely a e su ge y, he analgesic Me acam
(meloxicam) was used a a dose o 0.1mg‧kg-1 b.w. s.c.
once a day o h ee consecu i e days. Expe imen al ani-
mals ecei ed sys ema ic an ibio ic he apy (En o loxacin
15mg‧kg-1 b.w. once daily i.m. o 10days). A e sha -
ing he u o he back o he pig, an isepsis o he dono
a ea was pe o med wi h a 2.5% solu ion o po idone
iodine. Subsequen ly, using an elec ode ma ome (Zim-
me Biome ® Ai De ma ome, Zimme Biome , Indiana,
USA), a 0.20mm hin spli - hickness skin g a (STSG)
was emo ed wi h an a ea o 8 × 8cm in squa e a 6 si es
o planned skin de ec s. A sha p excision o an a ea meas-
u ing 8 × 8cm o ull- hickness skin (2–2.3cm dep h)
was pe o med a he si e o he emo ed skin g a s
(Fig.1). This was ollowed by he applica ion and ixa-
ion o he nanos uc u ed sca old and i s STSG co e .
The nanos uc u ed sca old consis s o Coll and CaOC-
based oam as he de mis laye ( hickness a ound 2mm)
and henano ibe s laye as a basal memb ane ( hickness
a ound 200 µm). The sca old on he igh was PDA-
coa ed (Fig.1e) and on he le was wi hou PDA. Anepi-
de mal g a wi hou sca old was used as a con ol. All
implan s we e ixed o he wound using a skin s aple
(Single-use Skin S aple B. B aun®, B. B aun, Ge many),
co e ed wi h g easy ulle and mule mois ened wi h Oc e-
nidine solu ion, and secu ed wi h a p essu e bandage.
Mic oso Excel and i s = RAND() unc ion we e used o
andomize he ypes o bioma e ials used in indi idual
ull- hickness skin de ec s.
The i s d essing change ollowed on he se en h pos -
ope a i e day, when he iabili y o he g a was e i ied.
The de ec s we e hen ied wi h a we mule and a g easy
ulle. His ological and immunohis ological samples we e
aken unde gene al anes hesia a indi idual s ages o
de ec healing on he 7 h and 14 h pos ope a i e day and
hen in he 3 d and 6 h pos ope a i e mon h. The pos -
incision de ec closu e was pe o med by di ec abso b-
able su u e.
His ological analysis andqPCR analysis o  issue samples
Fo maldehyde- ixed, pa a in-embedded issue samples
we e p ocessed o 2 his ological sec ions pe sample
and s ained wi h hema oxilin and eosin. Ligh mic os-
copy was used o e alua e he his ological images. Fo
pe o mance, mRNA was s abilized in issue samples
wi h an RNA La e ki (Quiagen, The Ne he lands). To al
RNA was isola ed using an RNeasy Mini Ki (Quiagen,
The Ne he lands) om 4 samples pe g oup and e e se
ansc ibed wi h he oligo-dT p ime and MMLV (In i -
ogen, USA) e e se ansc ip ase. P ime s o all genes
(e.g., IL1β, TNFα, TGFβ1, IL10) and he e e ence gene
(HPRT) we e used in p e ious publica ions o he eam
[44–46]. Based on he esul s ob ained om his ologi-
cal analyzes, o he genes (examina ion o genes associ-
a ed wi h cell dea h) we e also conside ed. Fo RT-PCR,
a Ligh Cycle 480 (Roche, Swi ze land) was used. Each
PCR eac ion consis ed o Quan iTec Syb G een mas-
e mix (Quiagen, The Ne he lands), 1μM o each p ime
and 1.0μL o cDNA in a o al olume o 10μL. Each sam-
ple was un in duplica e. The exp ession o a pa icula
gene was calcula ed as a mul iple o he exp ession o
he e e ence gene using he ollowing o mula: [1/(2C
GOI)]/[1/(2C HPRT)].
The mean exp ession o he HRT uni o p o-in lamma-
o y, an i-in lamma o y cy okines and g ow h ac o s was
compa ed using he T- es . Immunohis ological samples
wi h he Coll/CaOC, samples wi h he addi ion o PDA and
con ol g oup we e compa ed in each ime poin sepa a ely.
Page 7 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
Resul s
PDA in luences hes uc u e andmo phology
o  hebilaye
In his s udy, c oss-linked bilaye s and PDA-coa ed
c oss-linked bilaye s we e p epa ed and mo phologically
compa ed wi h non-c oss-linked bilaye s. Figu e2 shows
SEM isualiza ions o non-c oss-linked bilaye s and
PDA-coa ed c oss-linked bilaye s o show he signi ican
e ec o bo h in e en ions c oss-linking and PDA coa -
ing. The nano ib ous laye is placed on he su ace o he
sample (yellow a ow). A po ous sca old s uc u e can be
seen below i ( ed a ow). Figu e3 shows ade ailed SEM
isualiza ion o he adhesion be ween he nano ib ous
laye and he po ous oam. Figu e4 shows he nano i-
b ous s uc u e o he bilaye . Non-c oss-linked nano ib-
e s a e smoo h wi h andom ibe o ien a ion and wi h
ibe diame e in he ange o 370–500nm (Fig.4(NX)).
C oss-linked nano ibe s pa ially los hei ib ous s uc-
u e, and he ibe s a e al eady i mly a ached o each
o he , exhibi ing a mo e uni o m s uc u e (Fig.4(N)).
Non-co alen sel -assembly o  dopamine in c oss-link-
ing bilaye s p oduced PDA p ecipi a es deposi ed on
nano ibe s and almos con inuously co e ed he en i e
ib ous a ea (Fig. 4(N/PDA)). A sligh di e si y was
Fig. 1 C ea ion o 6 ull- hickness skin wounds 8 × 8 cm a scheme o wounds loca ion, b spli - hickness skin g a (STSG) dono si e, c ull- hickness
excision, d wound bed p epa a ion p io o sca old applica ion, e bilaye sca old made o collagen/oxidized cellulose oam wi h c oss-linked
nano ibe s—Coll/CaOC-N (le ) and bilaye sca old coa ed wi h polydopamine—Coll/CaOC-N/PDA ( igh ) applica ion di ec ly o wound bed,
applica ion o STSG on bilaye sca old, co e ing he ull- hickness skin de ec in one-s ep p ocedu e
Page 8 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
obse ed only o he Coll/CaOC-N/PDA sample. In his
case, PDA o med a con inuous ilm on he su ace o he
bilaye .
Figu e5a–d p esen s he e ec o PDA and c oss-link-
ing on o al bilaye hickness and po e sizes. He e, i is
e iden ha he c oss-linking p ocess as well as he PDA
addi ion lead o a dec ease in he bilaye hickness; he
ange o hickness is be ween 0.3 and 2.2mm (Fig.5a).
The hickness o hese samples was in mos cases hal
ha o he o iginal non-c oss-linked bilaye . Figu e5b
shows hepo e sizes o po ous oams wi hou he p es-
ence o nano ibe s, whe e hepo e sizes a e in he ange
o 50–250 µm and whe e PDA signi ican ly dec eases
hepo e sizes o all oams. The po e sizes o hepo ous
oams we e also measu ed om he c oss-sec ional a ea
o he bilaye s Fig.5c. A dec ease in hickness is ollowed
by a dec ease in hepo e sizes, while he po es change
shape o a hin ellipsoid. He e, he e is no signi icance
a e c oss-linking, only PDA p esen s signi ican esul s
in po e educ ion in some bilaye s. The po e size o he
Fig. 2 SEM isualiza ion o bilaye s made o po ous oam and nano ibe s. Collagen oam wi h c oss-linked nano ibe s is ep esen ed as Coll-N;
collagen/chi osan oam wi h c oss-linked nano ibe s is ep esen ed as Coll/Chi -N; and collagen/oxidized cellulose oam wi h c oss-linked
nano ibe s is ep esen ed as Coll/CaOC-N. The nex ma k ‘NX’ ep esen s he non-c oss-linked bilaye . The ma k ‘PDA’ ep esen s he polydopamine
coa ing on c oss-linked bilaye s. The nano ib ous laye is placed on he su ace o he sample (yellow a ow). A po ous sca old s uc u e can be seen
below i ( ed a ow)
Page 9 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
bilaye s was educed om 250 o 80µm o Coll-NX/
PDA and Coll-N/PDA, om 230 o 100 µm o Coll/
Chi -NX/PDA and Coll/Chi -N/PDA and om 120 o
60µm o Coll/CaOC-NX/PDA and Coll/CaOC-N/PDA,
espec i ely. The po e sizes on he su ace o henano ib-
e s a e in he ange o 0.7–4µm, in luenced by PDA and/
o c oss-linking (Fig.5d).
PDA in luences hes uc u e in eg i y o collagen
Figu e6a shows he ATR-FTIR spec a o he PDA and
non-PDA Coll, Coll/Chi and Coll/CaOC oamed sam-
ples, which consis o he cha ac e is ic abso p ions
ela ed o he O–H g oup and he N–H s e ching bonds
including he ypical collagen amide A a 3325 cm−1
and he amide B a 2924 cm−1. Amide Iis a ibu ed o
he s e ching ib a ions o he C=O g oups a 1600–
1800 cm−1. The ib a ions o he N–H bands and he
ib a ions o he C–N a e associa ed wi h amide II
(1470–1570 cm−1). The ib a ions o he C–N s e ching,
he N–H bending, and he ib a ions o he CH3 g oups
belong o amide III a 1250–1350 cm−1 [47]. Collagen
amide bonds A, B, I, II, and III a e con i med in all ypes
o PDA and non-PDA samples. The p esen ed abso p-
ion spec a look e y simila in he whole wa enumbe
ange. I is clea ha he amoun o indi idual biopoly-
me s (Coll, CaOC, Chi ) is la ge compa ed o he amoun
o PDA coa ing laye , so ha e y o en he bands o PDA
a e o e lap wi h he bands o collagen. The addi ion o
PDA has al eady been cha ac e ized by he C–O–H o
he ca echol g oups o PDA a 1410 cm−1 and he indole
ings isible a abou 1350 cm−1, which has also been
shown o depend on he concen a ion o PDA ( om
0.5 o 10mg.mL−1) [48–50]. The amoun o PDAadded
(2mg.mL−1) in ou expe imen s and he washing p ocess
du ing he p epa a ion o he samples led o a lowe inal
concen a ion o PDA in he samples ha jus coa ed
he biopolyme ibe s wi h hin laye . Figu e6b shows in
mo e de ail ep esen a i e spec a o he PDA and he
non-PDA coa ed Coll/Chi sample wi h a highligh ed
band o he PDA indole ing in he egion be ween 1230
Fig. 3 De ailed SEM isualiza ion o adhesion be ween po ous oam and nano ibe s. Collagen oam wi h c oss-linked nano ibe s is ep esen ed as
Coll-N; collagen/chi osan oam wi h c oss-linked nano ibe s is ep esen ed as Coll/Chi -N; and collagen/oxidized cellulose oam wi h c oss-linked
nano ibe s is ep esen ed as Coll/CaOC-N. The nex ma k ‘NX’ ep esen s he non-c oss-linked bilaye . The ma k ‘PDA’ ep esen s he polydopamine
coa ing on c oss-linked bilaye s. The nano ib ous laye is placed on he su ace o he sample (yellow a ow). A po ous sca old s uc u e can be seen
below i ( ed a ow)
Page 16 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
This exp ession was hen educed and was compa able
o ha o he non-PDA ma e ial. Ano he p o-in lam-
ma o y cy okine IL17 was signi ican ly highe a week 1
o hePDA ma e ial (p < 0.05), while mon h 6 showed
a signi ican dec ease in IL17 le el (p < 0.05), com-
pa ed o he non-PDA a ian . In con as , PDA was
ound o p omo e he elimina ion o in lamma ion, as
i suppo ed he exp ession o he an i-in lamma o y
molecule IL10 (mon h 3) du ing healing (p < 0.005).
Ano he cy okine TGFβ1 ( ans o ming g ow h ac o )
was ound o be signi ican ly exp essed in he healing
s ages o he ma e ial wi h added PDA (besides week
2). The le el o he las cy okine MMP9 (an enzyme o
he ma ix me allop o einase (MMP) amily) inc eased
Fig. 11 Li e-Dead assay o mu ine ib oblas cell line 3T3-A31 seeded on po ous oams on he 14 h expe imen al day. Collagen oam is Coll;
collagen/chi osan oam is Coll/Chi , and collagen/oxidized cellulose oam is Coll/CaOC. The ma k ‘PDA’ ep esen s an addi ional polydopamine
coa ing on po ous oams. The cy oplasm o li ing cells (g een luo escence) and dead cells ( ed luo escence). Scale ba 100 µm, objec i e 10 ×
Fig. 12 The cell dis ibu ion on he sca olds—day 14. Collagen oam is Coll; collagen/chi osan oam is Coll/Chi , and collagen/oxidized cellulose
oam is Coll/CaOC. The ma k ‘PDA’ ep esen s an addi ional polydopamine coa ing on po ous oams. Cy oplasmic memb anes (DiOC6[3], g een
signal), cell nuclei (p opidium iodide, ed signal). Scale ba 50 µm, objec i e 20 ×

Page 17 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
in he p esence o PDA o a signi ican ly highe le el a
week 3, compa ed o he con ol. Exp essions o MMP9
in he es s ages o wound healing we e compa able
be ween ma e ials and no signi ican ly di e en .
An ea ly in lamma ion is also e iden in he his o-
logical pic u es in Fig.16, he i s week a e implan a-
ion o Coll/CaOC-N and Coll/CaOC-N/PDA bilaye s
compa ed o he con ol. He e, asupe icial skin de ec
Fig. 13 Colo -coded dep h p ojec ion based on he signal o cells s ained wi h p opidium iodide and DiOC6(3) on day 14. Collagen oam is Coll;
collagen/chi osan oam is Coll/Chi , and collagen/oxidized cellulose oam is Coll/CaOC. The ma k ‘PDA’ ep esen s an addi ional polydopamine
coa ing on po ous oams. Dep h iew om 0 µm (blue) o 120 µm ( ed)
Fig. 14 Closu e o ull- hickness wounds a e implemen a ion o con ol g oup—na i e po cine skin a and bilaye made o collagen/oxidized
cellulose oam wi h c oss-linked nano ibe s—Coll/CaOC-N (b-le ) and bilaye coa ed wi h polydopamine—Coll/CaOC-N/PDA (b- igh ) o
6 mon hs
Page 18 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
is e iden and he de mis is in lammably cellula wi h
heimplan ed ma e ial. A e wo weeks, he e is s ill a
skin de ec on he su ace and he cellula g anula ion
issue is ich in capilla ies and ib oblas ic issue. The
con en o esidual implan ed ma e ial, mul inuclea ed
mac ophages, and ib oblas -like cells is p esen ed in he
su oundings. A e one mon h, he e is a ib ous cel-
lula sca wi h well-o ganized ib oblas o ma ion and
mild ch onic in lamma ion wi hou implan ma e ial.
The su ace de ec is al eady e-epi helialized. 6mon hs
o healing shows an o ganized ib ous sca wi h a lack
o ib oblas ic cells and capilla ies in he de mis. On he
wound su ace is he epide mis o he usual s uc u e
p esen ed.
Discussion
To da e, o all acellula sca olds, only human acellula
de mis p oduc s, such as clinically p o en AlloDe m and
De maMa ix appea o be he bes op ion in skin issue
[51, 52]. Implan a ion o acellula sca olds wi h cell cul-
u es o ib oblas s and/o ke a inocy es is associa ed wi h
eno mous cos s and di icul egula ions [53]. Cu en ly,
an example o a cell- ee ex acellula ma ix ep esen s
In eg a, made o Coll and chond oi in-6-sul a e wi h a sil-
icone backing [54]. This ma ix has also been seeded wi h
au ologous ib oblas s and ke a inocy es, bu i is no ye
comme cially a ailable. I is ime-de icien , as i equi es
3 o 4weeks o cul i a ion. Simila ly, 2weeks o ke a ino-
cy e cul i a ion is equi ed by simila ma e ial [55].
Fig. 15 Time dependence o mean exp ession o he HRT uni o p o-in lamma o y cy okines TNFα (a), IL1β (b), IL17 (c), MMP9 ( ),
an i-in lamma o y cy okines IL10 (d) and g ow h ac o TGFβ1 (e). Each panel ep esen s a speci ic ime o cy okines exp ession (1 week–6
mon hs) and implan ed ma e ial, whe e a bilaye made o collagen/oxidized cellulose c oss-linked wi h nano ibe s coa ed wi h polydopamine
is Coll/CaOC-N/PDA (o ange colo panel), a bilaye made o collagen/oxidized cellulose c oss-linked wi h nano ibe s is Coll/CaOC-N (g een
colo panel).( The panel o he con ol g oup (yellow colo panel) is he na i e po cine skin. S a is ical signi icance (*p < 0.05), (**p < 0.005),
(***p < 0.001)
Page 19 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
In his esea ch, bilaye acellula sca olds we e ab i-
ca ed and modi ied wi h PDA o enhance biomechanical
and biological p ope ies and o see he healing e ec s o
po cine skin, as well as in i o es s wi h mu ine ib o-
blas s. The nano ib ous laye unc ionally and s uc u -
ally ep esen s he BM, and he po ous oam ep esen s
he de mis laye . Fab ica ed nano ib ous BM is cha ac-
e ized by small po es and a e y hin hickness 0.2mm,
compa ed o he ab ica ed de mis laye , which is abou
2mm. I s main objec i e is o block bac e ia and ensu e
ha ib oblas s do no a el om he de mis pa o he
epide mis pa , as well as o suppo adhesion o a pos-
sible epide mal au og a . Po ous oam wi h la ge po es
and highe hickness ensu es ib oblas adhesion, p o-
li e a ion, nu ien suppo , and gene al illing o he
wound bed. PDA is ob ained by au o-polyme iza ion o
dopamine and has al eady shown some ad anced e ec s
on ma e ial p ope ies in issue enginee ing applica-
ions, mainly in e ms o mechanical and cellula pe -
o mance [56, 57]. In his wo k, he PDA imp o ed he
mechanical p ope ies o all bilaye s unde bo h d y and
hyd a ed condi ions. PDA esul ed in an inc ease in UTS
o app oxima ely 58%, 62%, and 35% o Coll-N/PDA,
Coll/Chi -N/PDA, and Coll/CaOC-N/PDA bilaye s,
espec i ely. In he hyd a ed s a e, PDA enhances he
i mness mainly in Coll/Chi -N/PDA (by 61%). The
hyd a ed en i onmen p o ided o he PDA-coa ed bilay-
e s wi h highe iscoelas ici y. One o he s iking ea-
u es o heal hy skin is i s abili y o e u n o no mal a e
being s e ched. The hyd a ed s a e and PDA signi ican ly
suppo ed ibe s elonga ion, which was conside ed ben-
e icial because he hyd a ed condi ions mimic he eal
issue en i onmen . The Coll/Chi -N/PDA bilaye was
s e ched by mo e han 80% in he hyd a ed s a e com-
pa ed o he d y s a e. Samples Coll-N/PDA and Coll/
CaOC-N/PDA elonga ed by mo e han 79–81% unde
hyd a ed condi ions, espec i ely. Adding PDA o an
al eady exis ing ne wo k o collagen and polysaccha ide
enhanced he mechanical igidi y o he ma e ial. PDA
con ains abundan hyd oxyl g oups o ca echoland ac i e
amino g oups, which can easily pene a e he ne wo k
and physically c oss-link wi h esidues o p o ein/cellu-
lose unc ional g oups and o m an in e pene a ed pol-
yme ne wo k. This ype o ne wo k shows signi ican ly
be e mechanical p ope ies han "o dina y" polyme
ne wo ks [58–60].
PDA has also slowed he enzyma ic deg ada ion,
while he swelling capaci y and po e size we e educed.
Fig. 16 His ological sec ions a a ious imes o issue ha es ing a e implan a ion o he con ol g oup-na i e po cine skin a, a bilaye o
collagen/oxidized cellulose c oss-linked wi h nano ibe s (Coll/CaOC-N) p esen ed as a well-o ganized sca issue b, collagen/oxidized cellulose
c oss-linked wi h nano ibe s coa ed wi h polydopamine (Coll/CaOC-N/PDA) he p esence o mac ophages and ib oblas -like cells in he 1s and
he 2nd week a e applica ion, collagen- ich issue wi h low p esence o ib oblas -like cells 6 mon hs a e applica ion c. Sec ions we e s ained
wi h H&E, pho og aphs a 200 × magni ica ion. Remnan s o he nano ibe laye s a e depic ed in a black a ow
Page 20 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
Al hough he po e s uc u e was educed, and he ma e-
ial became mo e igid, he Coll/CaOC/PDA showed he
highes p oli e a i e ac i i y be ween day 7 and day 14
and sa is ac o y me abolic ac i i y du ing he expe imen-
al pe iod. Oxidized cellulose oams Coll/CaOC and Coll/
CaOC/PDA, espec i ely, e ealed a o able p ope ies
o ini ial cell adhesion and p oli e a ion wi h possible
u he o ma ion o he con luen cell laye suppo ed
by PDA. G ea e sp ead o he cy oplasm and suppo ed
cell iabili y we e also obse ed in all samples a e PDA
coa ing. In ou expe imen s, we used he s a ic cul u e o
he sca olds. In he majo i y o 3D sca olds cul i a ed
unde s a ic condi ions, dead cells a e isible a e long-
e m cul i a ion, p obably due o slow medium di usion.
In addi ion, he physico-chemical p ope ies o he sca -
old su ace o sca old deg ada ion may nega i ely al e
cell adhesion, p oli e a ion, and iabili y o he cells. In
ou samples wi h PDA, signi ican ly (p < 0.05) inc eased
me abolic ac i i y o cells was obse ed. This is in good
ag eemen wi h Paccelli e  al. [61]. They epo ed an
inc eased we abili y o gellan gum hyd ogel coa ed wi h
PDA and subsequen ly inc eased celladhesion, sp ead-
ing, p oli e a ion, and ocal p o ein and cy oskele al
p o ein exp ession. Rega ding he su ace o he nano i-
b ous laye , PDA also changed i s su ace mo phology
and was able o c ea e di e en opologies depending on
he po ouscollagen/polysaccha ide oam, on which he
nano ibe s we e ab ica ed. Fo example, some un ea ed
esiduals o he sligh ly acidic CaOC o heColl/CaOC
oam could educe he polyme iza ion o dopamine and
c ea e a mo e homogeneous ilm o PDA compa ed o
ball-like s uc u es on he es o nano ibe s. This can
u he a ec he di usion o molecules.
In econs uc ing skin issue, i is essen ial o choose an
app op ia e wound in a p eclinical animal model. Mos
in i o s udies, including bilaye d essings, a e e alu-
a ed in small mammals, especially a s, mice, abbi s,
o guinea pigs due o hei cos and easy- o-handle p o-
cesses [62–64]. Recen ly, in i o s udies we e pe o med
wi h dopamine-modi ied ma e ials ha ha e also been
shown o heal wounds in hese small mammals, includ-
ing a s and mice [65–68]. To ou knowledge, only a ew
s udies ha e used he pig model, as a esul o expensi e
s udies and di icul ies due o i s size [69–71]. In addi-
ion, he e a e no s udies ha demons a e he e ec o
dopamine on po cine skin. Fu he mo e, wound heal-
ing in he pig model di e s om wound healing in small
mammals [72, 73]. Many au ho s ha e al eady sug-
ges ed ha pigs should be he p e e ed animal model
due o simila i ies be ween hei epide mis and de mis
wi h humans [74, 75]. In his wo k, a c ucial ques ion
ega ding PDA-coa ed bilaye ma e ial is i s in luence
on po cine skin du ing wound healing, especially an i-
in lamma o y beha io , as i is s ill li le known. The Coll/
CaOC-N/PDA bilaye was selec ed o he expe imen
and wascompa ed wi h i s non-PDA a ian Coll/CaOC-
N and he con ol g oup (na i e po cine skin). PDA sup-
po s he ea ly s ages o in lamma ion, since he le els o
he p o-in lamma o y cy okines TNFα, IL1β, and IL17
we e highly exp essed. P o-in lamma o y cy okines a e
among he i s ac o s ha a e p oduced in esponse
o wounds, as hey mus pa icipa e in he in lamma-
ion phase o wound healing wi h a mode a e immune
esponse only [76]. P ope le els o p o-in lamma o y
cy okines p e en in ec ion and accele a e no mal wound
healing [77]. The exp ession o he an i-in lamma o y
cy okine IL10 and he g ow h ac o TGFβ1 is highe in
he middle s ages o wound healing, due o PDA. PDA
also suppo ed MMP9, which could help in angiogenesis
and neo ascula iza ion p ocesses in he middle s age o
healing [78]. Theexp ession o all cy okines in his s udy
suppo ed he idea ha PDA igge s he ea ly s ages o
in lamma ion (1–3weeks) and p olongs in lamma ion a
he end o healing (6mon hs). A his poin , he o e ac i-
a ion o immune cells and hei p o ease p oduc s could
inhibi issue o ma ion.(1) An in lamma o y eac ion is
a dynamic p ocess wi h a epa a ion phase ollowed by
a ebuilding phase associa ed wi h mac ophages ac i a-
ionand he p oduc ion o TGFβ1 as well as MMPs. So
a , he e has been no di ec e idence o he ole o poly-
dopamine in mac ophageac i i y. The main an i-in lam-
ma o y unc ion o polydopamine is a ibu ed o he
abili y o elimina e eac i e oxygen species (ROS) [79].
The excessi e amoun o ROS p oduced by neu ophils a
he wound si e may des oy biological mac omolecules,
and hus i can cause a educ ion o he p oduc ion o
an i-in lamma o y molecules and con a y inc ease
he p oduc ion o p o-in lamma o y cy okines. PDA is
capable o cap u e elec ons and sca enge eac i e oxy-
gen species (ROS) ia i s ca echol g oups, which educe
in lamma ion and p omo e issue egene a ion [80, 81].
Ano he po en ial mechanism is al eady p oposed, based
on PDA ex ac s, which may ei he ac as a sca enge o
ROS o canac i a e an ioxidan p o ein HO-1and hus
inhibi he in lamma o y esponse.[82] Based on he
wound closu e and con ac ion in Fig.14 wi h wound
eco e y accompanied by hai g ow h, and less isual
sca ing om he PDA bilaye , i could be s a ed ha he
inc eased in lamma ion in he healing p ocess had no
e ec on he speed o wound eco e y. Al hough his o-
logical sec ions in Fig.16 e eal minimal di e ences in
his ological skin issue composi ion among na i e po -
cine skin, Coll/CaOC-N and Coll/CaOC-N/PDA, espe-
cially in he6 h mon h a e implan a ion, heau ho s
Page 21 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
o he s udy ind his a ou able. Acco ding o his ologi-
cal sec ions, he ma u a ion sca is ad anced in Coll/
CaOC-N and Coll/CaOC-N/PDA issue samples com-
pa ed o na i epo cine skin. The g oss obse a ions hen
a o ize Coll/CaOC-N/PDA o e Coll/CaOC-N samples,
as men ioned abo e. Elgha ably [83] used Coll gel o heal
ull- hickness excisional wounds o he po cine model
and also showed a obus in lamma o y esponse, which
esol ed in a imely manne ollowed by an imp o ed
p oli e a i e phase, angiogenic esul and pos -wound
issue emodeling. Middelkoop e al. [84] s udied Coll-
based ma e ials and syn he ic ma e ials in domes ic pigs
and poin ed o he disad an ages o d essing ea men s,
which we e no e ealed in in i o s udies. Philand i-
anos e  al. [85] showed ha a e implan ed a i icial
de mal subs i u es (In eg a, P oDe m, Renoskin, Ma i-
de m and Hyaloma ix) and he con ol g oup he e was
no di e en ial e ec on he con ac ion o ull- hickness
po cine wounds a e 2 and 6mon hs o healing. Ha -el
e al. [86] conduc ed a simila s udy be ween an elec o-
spun soy p o ein-based sca old (SPS) and Tegade m®
implemen ed in a ull- hickness wound in he pig model.
SPS exhibi ed be e e-epi helializa ion. Impo an ly, i
should be aken in o accoun ha s udies demons a ing
no isk o PDA deg ada ion p oduc s a e lacking. Jin e al.
[82] showed ha he PDA ex ac s we e mainly com-
posed o dopamine, quinine and PDA segmen s. These
deg adable p oduc s o PDA showed no cy o oxici y,
which is in good ag eemen wi h ou s udy.
Conclusions
This s udy e alua ed he e ec o dopamine coa ing in
a ully eso bable and acellula bilaye sca old made
o polysaccha ides and collagen. The PDA has a unique
posi ion in he c ea ion o biomechanically enginee ed
ma e ials as i signi ican ly changes he s eng h o he
bilaye and p omo es s abili y and iscoelas ici y due o
i s polyme ic ne wo k in e pene a ion. This also in lu-
ences swelling capaci y and po osi y, which is conside ed
desi able and bene icial o es o ing skin unc ion, since
dopamine also suppo s ib oblas iabili y and p oli -
e a ion. This esea ch con ibu es o he indings ha
dopamine may enhance he in lamma o y phase a he
beginning o wound healing in he domes ic pig model
and shows possible suppo in he expansion o g ow h
ac o and an i-in lamma o y cy okines in he middle and
la e s ages o wound healing. Dopamine shows no ox-
ici y du ing he healing p ocess, and he e o e he dopa-
mine-modi ied bilaye is sui able as implan able ma e ial.
Despi e he ema kable esul s o he in i o expe i-
men s, he ques ion emains whe he he u ili y o dopa-
mine in i o is no o e es ima ed, as i has been shown
o be esemble o he con ol g oup. The u u e pe spec-
i e on he u ili y o dopamine is mainly suppo ed by i s
ema kable esul s based on in i o expe imen s wi h
cells, which subs an ially p o e he po en ial o dopa-
mine. Howe e , i s mechanism o ac ion is s ill no com-
ple ely disco e ed and e en his s udy ailed o cla i y he
mechanism o dopamine, as well as i s deg adable p od-
uc s and possible in luence on he body. This ac ion a he
molecula le el needs o be disco e ed be o e any appli-
ca ion, which would also p o ide a be e unde s anding
o i s u u e inco po a ion in o bioma e ials, hus ob ain-
ing mo e p onounced in i o esul s.
Abb e ia ions
b.w Body weigh
CaOC Calcium sal o oxidized cellulose
Chi Chi osan
Coll Collagen
Coll/CaOC Collagen/oxidized cellulose oam
Coll/CaOC-N Collagen/oxidized cellulose bilaye ( oam + c oss-
linked nano ibe s)
Coll/CaOC-N/PDA PDA coa ed collagen/oxidized cellulose bilaye
( oam + c oss-linked nano ibe s)
Coll/CaOC-NX Collagen/oxidized cellulose bilaye ( oam + non-c oss-
linked nano ibe s)
Coll/CaOC/PDA Collagen/oxidized cellulose oam coa ed wi h PDA
Coll/Chi Collagen/chi osan oam
Coll/Chi -N Collagen/chi osan bilaye ( oam + c oss-linked
nano ibe s)
Coll/Chi -N/PDA PDA coa ed collagen/chi osan bilaye ( oam + c oss-
linked nano ibe s)
Coll/Chi -NX Collagen/chi osan bilaye ( oam + non-c oss-linked
nano ibe s)
Coll/Chi /PDA Collagen/chi osan oam coa ed wi h PDA
Coll-N Collagen bilye ( oam + c oss-linked nano ibe s)
Coll-N/PD PDA coa ed collagen bilye ( oam + c oss-linked
nano ibe s)
Coll-NX Collagen bilye ( oam + non-c oss-linked nano ibe s)
Coll/PDA Collagen oam coa ed wi h PDA
DMEM Dulbecco’s modi ied eagle medium
E. coli Esche ichia Coli
EDC/NHS N-(3-Dime hylaminop opyl)-N´-e hylca bodiimide
hyd ochlo ide/N-hyd oxysuccinimide
FGF2 Fib oblas g ow h ac o 2
Gel Gela in
i.m In amuscula
i.p In ape i oneal
i. In a enous
MRSA Me hicillin- esis an S. au eus
PBS Phospha e-bu e ed saline
PCL Poly(ε-cap olac one)
PDA Polydopamine
PEG Poly(e hylene glycol)
PLA Poly(lac ic acid)
PLGA Poly(lac ic-co-glycolic acid)
PVA Poly( inyl alcohol)
RhVEGF Recombinan human ascula endo helial g ow h
ac o
ROS Reac i e oxygen species
SEM Scanning elec on mic oscope
SEMIPE SEM image po e ex ac o
SeNPs Selenium nanopa icles
s.c. Subcu aneous

Page 22 o 24
Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80
Acknowledgemen s
No applicable.
Au ho con ibu ions
All au ho s con ibu ed o he s udy concep ion and design. KK w o e o iginal
d a , p epa ed ma e ials, pe o med he expe imen s and e alua ed mos
o he esul s. Au ho s who c i ically e iewed he manusc ip VP, EF, and LV.
Me hodology and supe ision o bioma e ial pa is a ibu ed o LV. Me h-
odology and supe ision o cells expe imen s in i o, analysis and da a col-
lec ion o VHB, and EF. Me hodology and supe ision o in i o expe imen is
a ibu ed o BL, MF, MK, and JH. Expe imen design o biomechanical analysis
is a ibu ed o PP. VP and LM cap u ed scanning elec on mic oscopy images.
ZP cap u ed his ological images. MC, MV, and JJ p o ided s a is ics o cy okine
exp ession. Concep ualiza ion—LV and BL. Funding acquisi ion—LV, BL, VP, EF
and MF. All au ho s ead and app o ed he inal manusc ip .
Funding
This esea ch was unded by he Minis y o Heal h o he Czech Republic
unde he p ojec no. 17-29874A and i s ex ended p ojec no. NU22-08–00454
as well as by he Minis y o Ag icul u e o he Czech Republic (RO0518), and
by EU Ho izon 2020 MSCA-RISE-2018 Resea ch and Inno a ion S a Exchange
P og amme, p ojec Ac iTOX unde he Ma ie Skłodowska-Cu ie g an ag ee-
men No 823981. CzechNanoLab p ojec LM2018110 unded by MEYS CR is
g a e ully acknowledged o he inancial suppo o he measu emen s/sam-
ple ab ica ion a CEITEC Nano Resea ch In as uc u e. All igh s ese ed.
A ailabili y o da a and ma e ials
Wi hou es ic ions.
Decla a ions
E hics app o al and consen o pa icipa e
The s udy was conduc ed acco ding o he guidelines o he Decla a ion
o Helsinki,and app o ed by he B anch Commission o Animal Wel a e
o he Minis y o Ag icul u e o he Czech Republic (pe mission numbe
21211/2017-MZE-17214 om 31 Ma ch 2017).
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Recei ed: 14 No embe 2022 Accep ed: 15 Feb ua y 2023
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