Accelular nanofbrous bilayer scafold intrapenetrated with polydopamine network and implemented into a full-thickness wound of a white-pig model afects infammation and healing process

Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80 h ps://doi.o g/10.1186/s12951-023-01822-5 RESEARCH © The Au ho (s) 2023. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons licence, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons licence and you in ended use is no pe mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his licence, isi h p:// c ea i eco mmons. o g/ licen ses/ by/4. 0/. The C ea i e Commons Public Domain Dedica ion wai e (h p:// c ea i eco mmons. o g/ publi cdoma in/ ze o/1. 0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed in a c edi line o he da a. 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 Re e ences 1. E on PA, Moldawe LL. Cy okines and wound healing: he ole o cy okine and an icy okine he apy in he epai esponse. J Bu n Ca e Rehabil. 2004;25(2):149–60. 2. We ne S, G ose R. Regula ion o wound healing by g ow h ac o s and cy okines. Physiol Re . 2003;83(3):835–70. 3. Flanagan M. A p ac ical amewo k o wound assessmen 1: physiology. B J Nu s. 1996;5(22):1391–7. 4. Xie J, Wille h SM, Li X, Macewan MR, Rade A, Sakiyama-Elbe SE, e al. The di e en ia ion o emb yonic s em cells seeded on elec ospun nano ibe s in o neu al lineages. Bioma e ials. 2009;30(3):354–62. 5. Kennedy KM, Bhaw-Luximon A, Jhu y D. Skin issue enginee ing: biologi- cal pe o mance o elec ospun polyme sca olds and ansla ional challenges. Regen Eng T ansl Med. 2017;3(4):201–14. 6. Ahmadi-Aghkand F, Gholizadeh-Ghaleh AS, Panahi Y, Da aee H, Go jikhah F, Gholizadeh-Ghaleh Aziz S, e al. Recen p ospec i e o nano ibe sca - olds ab ica ion app oaches o skin egene a ion. A i Cells Nanomed Bio echnol. 2016;44(7):1635–41. 7. Zhong SP, Zhang YZ, Lim CT. Tissue sca olds o skin wound healing and de mal econs uc ion. Wi es Nanomed Nanobi. 2010;2(5):510–25. 8. Demi A, Ce he E. Biopolyme s as wound healing ma e ials challenges and new s a egies. In: Pigna ello Rosa io, edi o . Bioma e ials applica ions o nanomedicine. London: InTech; 2011. 9. Shen YI, Song HHG, Papa AE, Bu ke JA, Volk SW, Ge ech S. Acellula hyd ogels o egene a i e bu n wound healing: ansla ion om a po - cine model. J In es De ma ol. 2015;135(10):2519–29. 10. Ruszczak Z. E ec o collagen ma ices on de mal wound healing. Ad D ug Deli Re . 2003;55(12):1595–611. 11. Meye M. P ocessing o collagen based bioma e ials and he esul ing ma e ials p ope ies. Biomed Eng Online. 2019;18(1):24. 12. Gaspa A, Moldo an L, Cons an in D, S anciuc AM, Sa bu Boe i PM, E i- mescu IC. Collagen-based sca olds o skin issue enginee ing. J Med Li e. 2011;4(2):172–7. 13. Voj o á L, Pa liňáko á V, Mucho á J, Kac inská K, B níko á J, Knoz M, e al. Healing and angiogenic p ope ies o collagen/chi osan sca olds en iched wi h hype s able FGF2-STAB p o ein: in i o, ex o o and in i o comp ehensi e e alua ion. Biomedicines. 2021;9(6):590. 14. Voj o á L, Zikmund T, Pa liňáko á V, Šalplach a J, Kalaso á D, P osecká E, e al. The 3D imaging o mesenchymal s em cells on po ous sca olds using high-con as ed x- ay compu ed nano omog aphy. J Mic osc. 2019;273(3):169–77. 15. Shephe d DV, Shephe d JH, Ghose S, Kew SJ, Came on RE, Bes SM. The p ocess o EDC-NHS c oss-linking o econs i u ed collagen ib es inc eases collagen ib illa o de and alignmen . APL Ma e . 2015;3(1):014902. 16. Yang C. Enhanced physicochemical p ope ies o collagen by using EDC/ NHS-c osslinking. Bull Ma e Sci. 2012;35(5):913–8. 17. Da idenko N, Schus e CF, Bax DV, Raynal N, Fa ndale RW, Bes SM, e al. Con ol o c osslinking o ailo ing collagen-based sca olds s abili y and mechanics. Ac a Bioma e . 2015;25:131–42. 18. Gu L, Shan T, Xuan Ma Y, Tay FR, Niu L. No el biomedical applica ions o c osslinked collagen. T ends Bio echnol. 2019;37(5):464–91. 19. Sun LP, Wang S, Zhang ZW, Wang XY, Zhang QQ. Biological e alua ion o collagen–chi osan sca olds o de mis issue enginee ing. Biomed Ma e . 2009;4(5):055008. 20. Ahmed S, Ik am S. Chi osan based sca olds and hei applica ions in wound healing. Achie Li e Sci. 2016;10(1):27–37. 21. Mucho á J, Hea nden V, Michlo ská L, Viš ejno á L, Za aďáko á A, Šme ko á K, e al. Mu ual in luence o selenium nanopa icles and FGF2- STAB® on biocompa ible p ope ies o collagen/chi osan 3D sca olds: in i o and ex o o e alua ion. J Nanobio echnology. 2021;19(1):103. 22. Do azilo á J, Mucho á J, Šme ko á K, Kočio á S, Di iš P, Kopel P, e al. Syne gis ic e ec o chi osan and selenium nanopa icles on biodeg ada- ion and an ibac e ial p ope ies o collagenous sca olds designed o in ec ed bu n wounds. Nanoma e ials. 2020;10(10):1971. 23. Meng X, Tian F, Yang J, He CN, Xing N, Li F. Chi osan and algina e poly- elec oly e complex memb anes and hei p ope ies o wound d essing applica ion. J Ma e Sci Ma e Med. 2010;21(5):1751–9. 24. Ka i VVSR, Kuppusamy G, Tallu i SV, Mannemala SS, Kollipa a R, Wadhwani AD, e al. Cu cumin loaded chi osan nanopa icles imp egna ed in o collagen-algina e sca olds o diabe ic wound healing. In J Biol Mac o- mol. 2016;93:1519–29. 25. No o na K, Ha elka P, Sopuch T, Kola o a K, Vosmanska V, Lisa V, e al. Cellulose-based ma e ials as sca olds o issue enginee ing. Cellulose. 2013;20(5):2263–78. 26. Zimni sky DS, Yu ksh o ich TL, Bychko sky PM. Syn hesis and cha ac e iza ion o oxidized cellulose. J Polym Sci A Polym Chem. 2004;42(19):4785–91. 27. Ma ina B, Ka eřina K, Milosla a R, Jan G, Ru a M. Oxycellulose: signi ican cha ac e is ics in ela ion o i s pha maceu ical and medical applica ions. Ad Polym Technol. 2009;28(3):199–208. 28. Š acho á V, Voj o á L, Pa liňák D, Voj ek L, Sedláko á V, Hy šl P, e al. No el elec ospun gela in/oxycellulose nano ibe s as a sui able pla o m o lung disease modeling. Ma e Sci Eng C. 2016;67:493–501. 29. Joseph B, Augus ine R, Kala ikkal N, Thomas S, Sean ie B, G ohens Y. Recen ad ances in elec ospun polycap olac one based sca olds o wound healing and skin bioenginee ing applica ions. Ma e Today Com- mun. 2019;19:319–35. Page 23 o 24 Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80 30. Ba ba isi M, Ma ino G, A menia E, Vincenzo Q, Rosso F, Po celli M, e al. Use o polycap olac one (PCL) as sca olds o he egene a ion o ne e issue. J Biomed Ma e Res A. 2015;103(5):1755–60. 31. Ding YH, Flo en M, Tan W. Mussel-inspi ed polydopamine o bio-su ace unc ionaliza ion. Biosu Bio ibol. 2016;2(4):121–36. 32. Tsai WB, Chen WT, Chien HW, Kuo WH, Wang MJ. Poly(dopamine) coa ing o sca olds o a icula ca ilage issue enginee ing. Ac a Bioma e . 2011;7(12):4187–94. 33. Sun X, Cheng L, Zhao J, Jin R, Sun B, Shi Y, e al. bFGF-g a ed elec ospun ib ous sca olds ia poly(dopamine) o skin wound healing. J Ma e Chem B. 2014;2(23):3636–45. 34. Ho CC, Ding SJ. S uc u e, p ope ies and applica ions o mussel-inspi ed polydopamine. J Biomed Nano echnol. 2014;10(10):3063–84. 35. Lou T, Leung M, Wang X, Chang JYF, Tsao CT, Sham JGC, e al. Bi-laye sca - old o chi osan/PCL-nano ib ous ma and PLLA-mic opo ous disc o skin issue enginee ing. J Biomed Nano echnol. 2014;10(6):1105–13. 36. Wang F, Wang M, She Z, Fan K, Xu C, Chu B, e al. Collagen/chi osan based wo-compa men and bi- unc ional de mal sca olds o skin egene a- ion. Ma e Sci Eng: C. 2015;52:155–62. 37. Lin HY, Chen SH, Chang SH, Huang ST. T i-laye ed chi osan sca old as a po en ial skin subs i u e. J Bioma e Sci Polym Ed. 2015;26(13):855–67. 38. Kilic Bek as C, Kimiz I, Sendemi A, Hasi ci V, Hasi ci N. A bilaye sca old p epa ed om collagen and ca boxyme hyl cellulose o skin issue enginee ing applica ions. J Bioma e Sci Polym Ed. 2018;29(14):1764–84. 39. Hasa s i S, Angspa A, A amwi P. Randomized clinical ial o he inno a- i e bilaye ed wound d essing made o silk and gela in: sa e y and e i- cacy es s using a spli - hickness skin g a model. J E id Based Comple Al e n Med. 2015;2015:1–8. 40. Ma W, Zhou M, Dong W, Zhao S, Wang Y, Yao J, e al. A bi-laye ed sca old o a poly(lac ic- co -glycolic acid) nano ibe ma and an algina e–gela in hyd ogel o wound healing. J Ma e Chem B. 2021;9(36):7492–505. 41. Yao W, Gu H, Hong T, Wang Y, Chen S, Mo X, e al. A bi-laye ed ubula sca old o e ec i e an i-coagulan in ascula issue enginee ing. Ma e Des. 2020;194: 108943. 42. Zhang S, Chen L, Jiang Y, Cai Y, Xu G, Tong T, e al. Bi-laye collagen/ mic opo ous elec ospun nano ibe sca old imp o es he os eochond al egene a ion. Ac a Bioma e . 2013;9(7):7236–47. 43. Slo iko á A, Voj o á L, Jančař J. P epa a ion and modi ica ion o collagen- based po ous sca old o issue enginee ing. Chem Pap. 2008;62:4. 44. Ky o a K, S epano a H, Rychlik I, Polansky O, Le a L, Sekelo a Z, e al. The esponse o po cine monocy e de i ed mac ophages and dend i ic cells o salmonella yphimu ium and lipopolysaccha ide. BMC Ve Res. 2014;10(1):244. 45. S epano a H, Pa lo a B, S ome o a N, Ond acko a P, S ejskal K, Slana I, e al. Di e en immune esponse o pigs o mycobac e ium a ium subsp. a ium and mycobac e ium a ium subsp. hominissuis in ec ion. Ve Mic obiol. 2012;159(3–4):343–50. 46. Viceno a M, Nech a alo a K, Chlebo a K, Kuce o a Z, Le a L, S epano a H, e al. E alua ion o in i o and in i o an i-in lamma o y ac i i y o biologi- cally ac i e phospholipids wi h an i-neoplas ic po en ial in po cine model. BMC Compl Al e n Med. 2014;14(1):339. 47. Ji Y, Yang X, Ji Z, Zhu L, Ma N, Chen D, e al. DFT-calcula ed IR spec um amide I, II, and III band con ibu ions o N -me hylace amide ine compo- nen s. ACS Omega. 2020;5(15):8572–8. 48. Zangmeis e RA, Mo is TA, Ta lo MJ. Cha ac e iza ion o polydopamine hin ilms deposi ed a sho imes by au oxida ion o dopamine. Langmui . 2013;29(27):8619–28. 49. Zhu S, Gu Z, Xiong S, An Y, Liu Y, Yin T, e al. Fab ica ion o a no el bio- inspi ed collagen–polydopamine hyd ogel and insigh s in o he o ma ion mechanism o biomedical applica ions. RSC Ad . 2016;6(70):66180–90. 50. Mallinson D, Mullen AB, Lamp ou DA. P obing polydopamine adhesion o p o ein and polyme ilms: mic oscopic and spec oscopic e alua ion. J Ma e Sci. 2018;53(5):3198–209. 51. Debels H, Hamdi M, Abbe on K, Mo ison W. De mal ma ices and bioengi- nee ed skin subs i u es. Plas Recons Su g Glob Open. 2015;3(1):e284. 52. Foley E, Robinson A, Maloney M. Skin subs i u es and de ma ology: a e iew. Cu De ma ol Rep. 2013;2(2):101–12. 53. Zhang Q, Wen J, Liu C, Ma C, Bai F, Leng X, e al. Ea ly-s age bilaye issue- enginee ed skin subs i u e o med by adul skin p ogeni o cells p oduces an imp o ed skin s uc u e in i o. S em Cell Res The . 2020;11(1):407. 54. Bello YM, Falabella AF, Eagls ein WH. Tissue-Enginee ed skin. Am J Clin De ma ol. 2001;2(5):305–13. 55. Bu le CE, O gill DP, Yannas IV, Comp on CC. E ec o ke a inocy e seeding o collagen-glycosaminoglycan memb anes on he egene a ion o skin in a po cine model. Plas Recons Su g. 1998;101(6):1572–9. 56. Hu Y, Dan W, Xiong S, Kang Y, Dhinaka A, Wu J, e al. De elopmen o collagen/polydopamine complexed ma ix as mechanically enhanced and highly biocompa ible semi-na u al issue enginee ing sca old. Ac a Bioma e . 2017;47:135–48. 57. Sha ma D, Jia W, Long F, Pa i S, Chen Q, Qyang Y, e al. Polydopamine and collagen coa ed mic o-g a ed polydime hylsiloxane o human mesenchy- mal s em cell cul u e. Bioac Ma e . 2019;4:142–50. 58. Fichman G, Schneide JP. Dopamine sel -polyme iza ion as a simple and powe ul ool o modula e he iscoelas ic mechanical p ope ies o pep ide-based gels. Molecules. 2021;26(5):1363. 59. Han X, Li M, Fan Z, Zhang Y, Zhang H, Li Q. PVA/Aga in e pene a ing ne wo k hyd ogel wi h as healing, high s eng h, an i eeze, and wa e e en ion. Mac omol Chem Phys. 2020;221(22):2000237. 60. Zhao D, Kim JF, Ignacz G, Pogany P, Lee YM, Szekely G. Bio-inspi ed obus memb anes nanoenginee ed om in e pene a ing polyme ne wo ks o polybenzimidazole/polydopamine. ACS Nano. 2019;13(1):125–33. 61. Pacelli S, Paolicelli P, Pe ali o S, Subham S, Gilmo e D, Va ani G, e al. In es iga ing he ole o polydopamine o modula e s em cell adhesion and p oli e a ion on gellan gum-based hyd ogels. ACS Appl Bio Ma e . 2020;3(2):945–51. 62. Magin CM, Neale DB, D inke MC, Willenbe g BJ, Reddy ST, la Pe le KM, e al. E alua ion o a bilaye ed, mic opa e ned hyd ogel d essing o ull- hick- ness wound healing. Exp Biol Med. 2016;241(9):986–95. 63. Eskanda inia A, Ke aya A, Agheb M, Ra ienia M, Amini Baghbado ani M, Na id S, e al. A no el bilaye wound d essing composed o a dense polyu- e hane/p opolis memb ane and a biodeg adable polycap olac one/gela in nano ib ous sca old. Sci Rep. 2020;10(1):3063. 64. Sie a-Sánchez Á, Fe nández-González A, Lizana-Mo eno A, Espinosa- Ibáñez O, Ma inez-Lopez A, Gue e o-Cal o J, e al. Hyalu onic acid bioma e ial o human issue-enginee ed skin subs i u es: P eclinical compa a i e in i o s udy o wound healing. J Eu Acad De ma ol Vene eol. 2020;34(10):2414–27. 65. Gong M, Yan F, Yu L, Li F. A dopamine-me hac yla ed hyalu onic acid hyd o- gel as an e ec i e ca ie o s em cells in skin egene a ion he apy. Cell Dea h Dis. 2022;13(8):738. 66. Lee SY, Jeon S, Kwon YW, Kwon M, Kang MS, Seong KY, e al. Combina o ial wound healing he apy using adhesi e nano ib ous memb ane equipped wi h wea able LED pa ches o pho obiomodula ion. Sci Ad . 2022;8:15. 67. Zheng Z, Li M, Shi P, Gao Y, Ma J, Li Y, e al. Polydopamine-modi ied collagen sponge sca old as a no el de mal egene a ion empla e wi h sus ained elease o pla ele - ich plasma o accele a e skin epai : a one-s ep s a egy. Bioac Ma e . 2021;6(8):2613–28. 68. Yazdi MK, Za e M, Khodadadi A, Seidi F, Sajadi SM, Za in aj P, e al. Polydopamine bioma e ials o skin egene a ion. ACS Bioma e Sci Eng. 2022;8(6):2196–219. 69. Sea on M, Hocking A, Gib an NS. Po cine models o cu aneous wound heal- ing. ILAR J. 2015;56(1):127–38. 70. Tapking C, Popp D, B anski LK. Pig model o es issue-enginee ed skin. In: Bö che -Habe ze h Sophie, Biede mann Thomas, edi o s. Skin issue enginee ing: me hods and p o ocols. New Yo k: Sp inge ; 2019. p. 239–49. 71. S icke -K ong ad A, Shoemake CR, Boucha d GF. The minia u e swine as a model in expe imen al and ansla ional medicine. Toxicol Pa hol. 2016;44(4):612–23. 72. Sulli an TP, Eagls ein WH, Da is SC, Me z P. The pig as a model o human wound healing. Wound Repai Regen. 2001;9(2):66–76. 73. Sie a-Sánchez Á, Kim KH, Blasco-Mo en e G, A ias-San iago S. Cellula human issue-enginee ed skin subs i u es in es iga ed o deep and di - icul o heal inju ies. NPJ Regen Med. 2021;6(1):35. 74. Debee S, le Luduec JB, Kaise lian D, Lau en P, Nicolas JF, Dubois B, e al. Compa a i e his ology and immunohis ochemis y o po cine e sus human skin. Eu J De ma ol. 2013;23(4):456–66. 75. Khiao In M, Richa dson KC, Loewa A, Hed ich S, Kaessmeye S, Plendl J. His- ological and unc ional compa isons o ou ana omical egions o po cine skin wi h human abdominal skin. Ana His ol Emb yol. 2019;48(3):207–17. 76. Xiao T, Yan Z, Xiao S, Xia Y. P oin lamma o y cy okines egula e epide mal s em cells in wound epi helializa ion. S em Cell Res The . 2020;11(1):232. Page 24 o 24 Kac inskáe al. Jou nal o Nanobio echnology (2023) 21:80 • as , con enien online submission • ho ough pee e iew by expe ienced esea che s in you ield • apid publica ion on accep ance • suppo o esea ch da a, including la ge and complex da a ypes • gold Open Access which os e s wide collabo a ion and inc eased ci a ions maximum isibili y o you esea ch: o e 100M websi e iews pe yea • A BMC, esea ch is always in p og ess. Lea n mo e biomedcen al.com/submissions Ready o submi you esea ch Ready o submi you esea ch ? Choose BMC and bene i om: ? Choose BMC and bene i om: 77. Aki a S. Wound epai egen: mechanisms, signaling. In J Mol Sci. 2019;20(24):6328. 78. Caley MP, Ma ins VLC, O’Toole EA. Me allop o einases and wound healing. Ad Wound Ca e. 2015;4(4):225–34. 79. Zhao H, Zeng Z, Liu L, Chen J, Zhou H, Huang L, e al. Polydopamine nano- pa icles o he ea men o acu e in lamma ion-induced inju y. Nanoscale. 2018;10(15):6981–91. 80. Li Y, Yang L, Hou Y, Zhang Z, Chen M, Wang M, e al. Polydopamine-medi- a ed g aphene oxide and nanohyd oxyapa i e-inco po a ed conduc i e sca old wi h an immunomodula o y abili y accele a es pe iodon al bone egene a ion in diabe es. Bioac Ma e . 2022;18:213–27. 81. Zheng B, Deng G, Zheng J, Li Y, Wang B, Ding X, e al. Sel -polyme ized polydopamine-based nanopa icles o acu e kidney inju y ea men h ough inhibi ing oxida i e damages and in lamma o y. In J Biochem Cell Biol. 2022;143:106141. 82. Jin L, Yuan F, Chen C, Wu J, Gong R, Yuan G, e al. Deg ada ion P oduc s o polydopamine es ained in lamma o y esponse o LPS-s imula ed mac- ophages h ough media ion TLR-4-MYD88 dependen signaling pa hways by an ioxidan . In lamma ion. 2019;42(2):658–71. 83. Elgha ably H, Ganesh K, Dicke son J, Khanna S, Abas M, Das Gha ak P, e al. A modi ied collagen gel d essing p omo es angiogenesis in a p e- clinical swine model o ch onic ischemic wounds. Wound Repai Regen. 2014;22(6):720–9. 84. Middelkoop E, an den Bogae d AJ, Lamme EN, Hoeks a MJ, B andsma K, Ul ich MMW. Po cine wound models o skin subs i u ion and bu n ea - men . Bioma e ials. 2004;25(9):1559–67. 85. Philand ianos C, And ac-Meye L, Mo don S, Feue s ein JM, Saba ie F, Ve an J, e al. Compa ison o i e de mal subs i u es in ull- hickness skin wound healing in a po cine model. Bu ns. 2012;38(6):820–9. 86. el Ha -el Y, Ge s enhabe JA, B odsky R, Huneke RB, Lelkes PI. Elec ospun soy p o ein sca olds as wound d essings: enhanced eepi helializa ion in a po cine model o wound healing. Wound Med. 2014;5:9–15. Publishe ’s No e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in pub- lished maps and ins i u ional a ilia ions.