1
S uc u e-p ope ies ela ionship o chi osan/collagen ilms wi h po en ial
o biomedical applica ions
Mi eia Andonegia, Ke in Las He asb, Edo a San os-Vizcaínob,c, Manoli
Iga uab,c, Rosa Ma ia He nandezb,c, Ko o de la Cabaa*, Ped o Gue e oa*
aBIOMAT Resea ch G oup, Uni e si y o he Basque Coun y (UPV/EHU),
Escuela de Ingenie ía de Gipuzkoa, Plaza de Eu opa 1, 20018 Donos ia-San
Sebas ián, Spain.
bNanoBioCel G oup, Labo a o y o Pha maceu ics, School o Pha macy,
Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la Uni e sidad 7,
01006 Vi o ia-Gas eiz, Spain.
cBiomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and
Nanomedicine (CIBER-BBN). Vi o ia-Gas eiz, Spain.
*Co esponding au ho s
Ko o de la Caba
BIOMAT esea ch g oup
Uni e si y o he Basque Coun y (UPV/EHU)
Escuela de Ingenie ía de Gipuzkoa
Plaza de Eu opa 1, 20018 Donos ia-San Sebas ián (Spain)
E-mail: [email p o ec ed]
Ped o Gue e o
BIOMAT esea ch g oup
Uni e si y o he Basque Coun y (UPV/EHU)
Escuela de Ingenie ía de Gipuzkoa
Plaza de Eu opa 1, 20018 Donos ia-San Sebas ián (Spain)
E-mail: [email p o ec ed]
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Ca bohyd a e Polyme s
237 : (2020) // A icle ID 116159, which has been published in inal o m a h ps://doi.o g/10.1016/
j.ca bpol.2020.116159. © 2020 Else ie unde CC BY-NC-ND license (h p://c ea i ecommons.o g/
licenses/by-nc-nd/4.0/)
2
Abs ac 1
Chi osan/collagen ilms we e de eloped and cha ac e ized in o de o assess 2
he sui abili y o hese ilms o biomedical applica ions. Hence, 3
physicochemical, he mal, ba ie and mechanical p ope ies we e analyzed and 4
ela ed o he ilm s uc u e, which showed he p e alence o he iple helix o 5
na i e collagen a e he addi ion o chi osan. Fu he mo e, collagen ibe 6
diame e changed om 3.9 ± 0.6 μm, o collagen ilms wi hou chi osan, o 1.8 7
± 0.5 μm, o collagen ilms wi h low molecula weigh chi osan. These esul s 8
sugges ed in e ac ions be ween collagen and chi osan molecules, as obse ed 9
by Fou ie ans o m in a ed (FTIR) analysis. Rega ding ilm ba ie p ope ies, 10
chi osan/collagen ilms showed a wa e apo ansmission a e a ound 1174 11
g∙m-2∙day-1, sui able o biomedical applica ions such as wound healing. 12
Addi ionally, biological es s con i med ha he chi osan/collagen ilms 13
de eloped a e sui able o biomedical applica ions. 14
Keywo ds: chi osan, collagen, ilm, iple helix, bioma e ial. 15
3
1. In oduc ion 16
Skin is he la ges o gan o human body and i is he i s line o p o ec ion 17
agains en i onmen al exposu e, p e en ing un egula ed loss o body luid. 18
Annually, millions o people su e ed cu aneous wounds caused by physical and 19
chemical ac o s o diseases (P oksch, B andne , & Jensen, 2008). Those 20
wounds migh be exposed o in ec ion by pa hogenic bac e ia so, wound healing 21
is an essen ial p ocess o emodeling inju ed issues (Boa eng, Ma hews, 22
S e ens, & Eccles on, 2008). In ac , cu aneous wound healing can be di ided 23
in o ou o e lapping phases, haemos asis, in lamma ion, p oli e a ion and 24
emodeling, which in ol e di e se ypes o cells (Chen e al., 2019; Dabi i, 25
Dams e e , & Phillips, 2016). Any dis u bance in hese phases causes 26
p olonged healing, inc easing he isk o in ec ion (Li e al., 2019). The e o e, 27
di e en wound d essings, such as co on gauze d essings (F ykbe g & Bank, 28
2015), nano ib ous d essings (Ga cia-O ue e al., 2019), human amnio ic 29
memb ane (Zelen, Se ena, Denozie e, G., & Fe e ol , 2013), and 30
polysaccha ide-based d essings (Ding, Deng, Du, Shi, & Wang, 2014; Khalid, 31
Khan, UI-Islam, Khan, & Wahid, 2017; Qin, 2008), ha e been in es iga ed wi h 32
he aim o assis and p omo e he cu aneous wound healing p ocess, 33
main aining op imum condi ions o he ees ablishmen o he damaged issue. 34
In his con ex , he use o biopolyme s such as collagen and chi osan has been 35
sugges ed as an al e na i e ea men o wounds, aking in o accoun ha an 36
ideal wound d essing should be non-alle genic and non- oxic, p ese e mois 37
en i onmen , p omo e e ec i e oxygen exchange, p o ec he wound agains 38
mic obial o ganisms and abso b wound exuda es (Pe ez-Puyana, Jiménez-39
Rosado, & Gue e o, 2019) 40
4
In his ega d, collagen is a ib ous p o ein ha plays an impo an ole in issue 41
healing, p o iding he biological mic oen i onmen o cell g ow h and 42
suppo ing cell a achmen , mig a ion, and p oli e a ion (So kio e al., 2015). A 43
he same ime, i is biocompa ible, biodeg adable and low immunogenic 44
(Gopina h e al., 2004). Fo hese easons, collagen has become a p omising 45
biopolyme in egene a i e medicine (Pawelec, Bes , & Came on, 2016). 46
Howe e , modi ica ion o c oss-linking, using aldehydes (Tonndo , Aibibu, & 47
Che i , 2020) o ca bodiimides (Beghe o, Ga o, Conca, Ba della, & Sc i an i, 48
2019), is essen ial o issue enginee ing, since he deg ada ion a e and 49
mechanical s abili y o single collagen a e insu icien (Li e al., 2019). 50
Ne e heless, he c osslinking agen s used equen ly cause cy o oxici y 51
p oblems (Bae, Wang, & Ku isawa, 2013; Pe ez-Puyana, Jiménez-Rosado, & 52
Gue e o, 2019) and, hus, new c osslinking s a egies mus be assessed. 53
Rega ding chi osan, his is a semi-c ys alline polysaccha ide ob ained by 54
deace yla ion o chi in and i is widely used as a bioma e ial due o i s 55
biocompa ibili y, biodeg adabili y, non- oxici y, an imic obial and an i ungal 56
ac i i y (Ahmed & Ik am, 2016). Chi osan also has a bene icial e ec as a 57
wound healing p omo e , imp o ing he unc ions o ib oblas s, mac ophages 58
and in lamma o y cells (Rezaii, O yan, & Ja e i, 2019). In he las decades, 59
chi osan has also been s udied o cu aneous wound healing in he o m o 60
memb anes, colloidal solu ions o sponges (Bui, Pa k, & Lee, 2017; Elsabee & 61
Abdou, 2013). Howe e , he e ec o pu e chi osan on p omo ing cell 62
p oli e a ion is limi ed, and i s biodeg adabili y depends on i s deace yla ion 63
deg ee (Gamiz-Gonzalez e al., 2017). 64
5
Conside ing he unc ional p ope ies o collagen and chi osan, hei 65
combina ion could lead o composi es wi h enhanced mechanical p ope ies, 66
posi i e e ec s on cell p oli e a ion, and con olled deg ada ion eloci y. 67
Al hough collagen-chi osan composi es ha e been p e iously in es iga ed o 68
di e en issue enginee ing applica ions (Ra e y e al., 2016; Si, Yang, Xing, 69
Yang, & Shan, 2019; Yan e al., 2019), he e a e ew epo s ela ed o collagen-70
chi osan composi es o cu aneous wound healing and hose wo ks e e ed o 71
gels (Li e al., 2019) and sca olds (Rezaii, O yan, Ja e i, 2019). In his con ex , 72
he aim o his wo k is ocused on he de elopmen o ilms based on na i e 73
collagen, assessing he e ec o he inco po a ion o chi osan wi h high and low 74
molecula weigh on physicochemical, he mal, ba ie , mechanical and 75
biological p ope ies o he esul ing composi e ilms and ela ing hese 76
p ope ies o he ilm s uc u e. I is wo h no ing ha , in con as o o he wo ks 77
on collagen ilms, in which c osslinke s such as 1-e hyl-3-(3-dime hyl 78
aminop opyl) ca bodiimide (EDC) and N-hyd oxysuccinimide (NHS) a e added 79
(Soc a es e al., 2016), in his wo k he e was no need o inco po a e any 80
c osslinke in o he ilm o ming o mula ion due o he p e alence o he iple 81
helix s uc u e o na i e collagen o con ol and chi osan-inco po a ed ilms. 82
2. Ma e ials and me hods 83
2.1. Ma e ials 84
Bo ine collagen om immings and he spli ing-de i ed esidues, wi h 33% 85
glycine, 22% imino acids, 12% p oline, 11% alanine and 10% hyd oxyp oline, 86
was supplied by Tene ias Omega (Spain). Low molecula weigh (LMW, 190 87
kDa) chi osan (ba ch MKBB9037) and high molecula weigh (HMW, 375 kDa) 88
chi osan (ba ch MKBC0059) wi h a deg ee o deace yla ion highe han 75% 89
6
we e p o ided by Sigma-Ald ich (Spain). Glyce ol wi h a pu i y o 99.01% and 90
analy ical g ade, used as plas icize , was supplied by OPPAC P oduc s S.A 91
(Spain). 92
2.2. P epa a ion o ilms 93
The ea men s o bo ine skin o ob ain na i e collagen we e ca ied ou 94
acco ding o he me hod o Andonegi e al. (2020). Collagen ilms wi h 30 w . % 95
(on collagen d y basis) LMW chi osan and HMW chi osan we e p epa ed by 96
solu ion cas ing. Fi s ly, collagen skins we e ea ed wi h NaOH 1 M a oom 97
empe a u e o 12 h. These samples we e neu alized wi h phospha e bu e 98
saline (PBS). Collagen was g inded and eeze-d ied in o de o acili a e he 99
subsequen p ocessing. A e wa ds, 1.5 g chi osan we e dissol ed in 100 mL o 100
0.5 M ace ic acid unde con inuous s i ing o 30 min and hen, 5.0 g collagen 101
and 20 w . % glyce ol (on collagen d y basis) we e added. The blend was 102
main ained unde mechanical s i ing o 3 h and hen, pou ed in o Pe i dishes 103
and le d ying a oom empe a u e o ob ain he ilms. Con ol samples we e 104
p epa ed wi hou chi osan. 105
2.3. Mois u e con en (MC) and mass loss (ML) 106
To de e mine he MC o ilms, samples we e weighed (w0) and hen eeze-107
d ied. A e ha , samples we e eweighed (w1) and MC was calcula ed as: 108
MC ሺ%ሻ=w0-w1
w0
x 100 109
Mass loss alues we e calcula ed using d ied specimens. Rec angula ilm 110
pieces (w1) we e imme sed in o PBS (pH 7.4) o 5 days and hen, he samples 111
we e eweighed (w2). The mass loss o h ee specimens o each sample was 112
calcula ed as: 113
7
Mass loss ሺ%ሻ=w1-w2
w1
x 100 114
2.4. Swelling 115
Rec angula pieces we e weighed (wi) and hen, imme sed in o PBS in o de o 116
main ain he solu ion pH cons an . Samples we e weighed a e imme sion in o 117
PBS o speci ic imes (w ), un il cons an alues we e achie ed. The swelling 118
was calcula ed as ollows: 119
%1 00
i
ww
Swelling x
w
120
2.5. Fou ie ans o m in a ed (FTIR) spec oscopy 121
Fou ie ans o m in a ed (FTIR) spec a we e eco ded on a Nicole 380 FTIR 122
spec ome e equipped wi h ho izon al a enua ed o al e lec ance (ATR) c ys al 123
(ZnSe). A o al o 32 scans we e made a 4 cm-1 esolu ion. All spec a we e 124
smoo hed using he Sa i zky–Golay unc ion. Second-de i a i e spec a o he 125
amide egion we e used a peak posi ion guides o he cu e i ing p ocedu e, 126
using O iginP o 2019b so wa e. 127
2.6. The mo-g a ime ic analysis (TGA) 128
The he mal s abili y o samples was measu ed by using he Me le Toledo 129
TGA/SDTA 851 he mo-balance. Dynamic scans om 25 o 750 ºC we e ca ied 130
ou a a cons an a e o 10 ºC/min unde ni ogen a mosphe e (10 mL N2/min) 131
o a oid he mo-oxida i e eac ions. 132
2.7. Di e en ial Scanning Calo ime y (DSC) 133
DSC measu emen s we e pe o med on a Me le Toledo DSC 822. Samples o 134
a ound 3 mg we e subjec ed o a hea ing amp om 25 o 250 ºC a a a e o 10 135
8
ºC/min unde ni ogen a mosphe e o a oid oxida i e eac ions. Sealed 136
aluminum pans we e used o p e en mass loss du ing he expe imen . 137
2.8. X- ay di ac ion (XRD) 138
XRD was pe o med wi h a PANaly ical Xpe PRO di ac ion uni , ope a ing a 139
40 kV and 40 mA. The adia ion was gene a ed om a Cu-Kα (λ= 1.5418 Å) 140
sou ce. The di ac ion da a we e collec ed om 2θ alues om 2 o 50 º, whe e 141
θ is he angle o incidence o he X- ay bean on he sample. 142
2.9. Scanning elec on mic oscopy (SEM) 143
A Hi achi S-4800 scanning elec on mic oscope was used. P io o obse a ion, 144
samples we e moun ed on a me al s ub wi h double-side adhesi e ape and 145
coa ed unde acuum wi h gold, using a JEOL ine-coa ion spu e JFC-1100, 146
in an a gon a mosphe e. All samples we e examined using an accele a ing 147
ol age o 15 kV. Fo he collagen ibe diame e e alua ion (ᴓ) SEM images 148
we e analyzed by ImageJ so wa e. 149
2.10. Ul a iole - isible (UV- is) spec oscopy 150
The ligh -ba ie p ope ies o ilms we e de e mined by measu ing hei ligh 151
abso p ion a wa eleng hs anging om 200 nm o 800 nm, using a UV-Jasco 152
V-630 spec opho ome e . 153
2.11. Wa e apo ansmission a e (WVTR) 154
WVTR was de e mined in a con olled humidi y en i onmen chambe , using a 155
Lab hink PERME™ W3/0120 wa e apo ansmission a e es e . Ci cles o 156
7.40 cm diame e , wi h a es a ea o 33 cm2, we e cu . The es was ca ied ou 157
acco ding o ASTM E96-00 (ASTM, 2000). The se up was subjec ed o a 158
9
empe a u e o 38 °C and a ela i e humidi y o 90%. WVTR alues we e 159
calcula ed by he ollowing exp ession: 160
WVTR ൬g
day cmଶ൰=G
x A 161
whe e G is he change in weigh (g), is ime (day), and A is he es a ea (cm2). 162
2.12. Wa e con ac angle (WCA) 163
Wa e con ac angle measu emen s we e pe o med using a Da aPhysics OCA 164
20 con ac angle sys em. A 3 μL d ople o dis illed wa e was placed on ilm 165
su ace o es ima e i s hyd ophobic o hyd ophilic cha ac e . The image o he 166
d op was cap u ed using SCA20 so wa e. 167
2.13. Mechanical p ope ies 168
Tensile s eng h (TS) and elonga ion a b eak (EB) we e measu ed a oom 169
empe a u e, using a MTS Insigh 10 elec omechanical es ing sys em (MTS, 170
Spain), o d y and we specimens. We samples we e hyd a ed o 2 h in o 200 171
mL o dis illed wa e . Tensile es s we e pe o med acco ding o ASTM D 638-172
03 s anda d (ASTM, 2003). Specimens o each sample we e cu in o bone-173
shaped samples o 4.75 mm × 22.25 mm and he c osshead a e was 174
1 mm/min. 175
2.14. Deg ada ion s udies 176
Fo he de e mina ion o he weigh loss due o hyd oly ic and enzyma ic 177
p ocesses, ilms (n=3) wi h a diame e o 8 mm and an a ea o 110 m2 we e 178
weighed and subsequen ly imme sed in o di e en solu ions. 8 mm diame e 179
disks we e used due o he ease o handling and he sui abili y o es ing in 24-180
well pla es, as shown in p e ious in i o s udies (Ga cia-O ue e al., 2019). PBS 181
16
Conce ning DSC measu emen s, all samples exhibi ed h ee endo he mic 314
peaks, as shown in Figu e 2B. The peak a ound 90 ºC was ela ed o he ee 315
and bound wa e elease (Kaczma ek, Sionkowska, & Skopinska-Wisniewska, 316
2018). The dena u a ion empe a u e was associa ed o he endo he mic peak 317
a ound 150 ºC (Chak apani, Gnanamani, Gi ide , Madhusoo hanan, & Seka an, 318
2012). The empe a u e a which he maximum o his peak appea ed inc eased 319
wi h he addi ion o chi osan, and he highes alue was ound o he ilms wi h 320
LMW chi osan. This ac would con i m ha collagen-chi osan in e ac ions would 321
be a o ed o he chi osan wi h lowe molecula weigh , as p e iously shown by 322
swelling esul s. Finally, he hi d endo he mic peak appea ed be ween 210 and 323
250 ºC, and i was associa ed o con o ma ional changes om he iple helix 324
s uc u e o collagen o andom coil (Bozec & Odlyha, 2011). The empe a u e 325
co esponding o he maximum o his peak inc eased wi h he addi ion o 326
chi osan due o he exis ence o in e ac ions be ween collagen and chi osan, as 327
obse ed by FTIR esul s. In pa icula , DSC e ealed he cha ac e is ic 328
collagen-collagen and collagen-wa e in e ac ions o con ol ilms. In he ilms 329
wi h chi osan, hese in e ac ions a e pa ially eplaced by in e ac ions be ween 330
collagen and chi osan, indica ing ha he o ma ion o hyd ogen bonds be ween 331
collagen and chi osan compe ed wi h hyd ogen bonding be ween collagen 332
chains. Simila esul s ha e been ound o collagen- ucoidan ilms, showing he 333
inc ease o he mal s abili y by he inco po a ion o polysaccha ides in o 334
collagen ilm o ming o mula ions (Pe umal e al., 2018). 335
3.3. Mo phological p ope ies 336
The changes showed abo e equi e in o ma ion abou he s uc u e o 337
chi osan/collagen ilms, hus XRD and SEM analyses we e ca ied ou . All he 338
17
ilms exhibi ed XRD pa e ns o amo phous ma e ials (Figu e 3A). The small
339
e lec ion peak a 10° is a ibu ed o he chi osan c ys al o m (Lece a, Peñalba,
340
A ana, Gue e o, & de la Caba, 2015) and
he peak a ound 7º is ela ed o he
341
iple helix s uc u e o collagen (Valencia, Luciano, Lou enço, Bi an e, &
342
Sob al, 2019). Collagen molecules gene ally assemble in o ib ils and hen o m
343
collagen ibe s by in e molecula c oss-linking, leading o he cha ac e is ic
344
hie a chical in e wo en s uc u e o collagen. Acco ding o XRD esul s, he
345
addi ion o chi osan did no cause any ele an change, indica ing ha he
346
helical s uc u e o collagen was main ained a e chi osan addi ion, in
347
consis ence wi h FTIR esul s. Addi ionally, a b oad band appea ed a ound 20º,
348
associa ed o he di use sca e ing o collagen ibe s, indica ing he amo phous
349
s uc u e o he ilms (Zou e al., 2017). As can be seen, all samples showed
350
simila XRD pa e ns wi h a sligh inc ease in he in ensi y o he band a 20º o
351
he ilms wi h HMW chi osan, indica i e o he inc ease in he s uc u al o de .
352
353
Figu e 3. Mo phological p ope ies o chi osan/collagen ilms: A) XRD pa e ns
354
and SEM images o su aces o B) con ol ilms, C) collagen ilms wi h LMW
355
chi osan, and D) collagen ilms wi h HMW chi osan.
356
To u he asses he changes caused by he addi ion o chi osan in collagen
357
ilms, SEM analysis was ca ied ou and he images o ilm su ace a e shown in
358
Figu e 3B, C and D. SEM mic og aphs exhibi ed he ib il s uc u e o collagen
359
o all he ilms, showing he collagen pe iodici y co esponding o na i e
360
18
collagen (Soc a es e al., 2019). Wi h he addi ion o chi osan, he su ace was 361
oughe and mo e ibe s could be obse ed, especially o collagen ilms wi h 362
HMW chi osan. Fu he mo e, a signi ican (P < 0.05) dec ease o collagen ibe 363
diame e was obse ed om 3.9 ± 0.6 μm o con ol ilms o 1.8 ± 0.5 μm and 364
1.9 ± 0.5 μm o he ilms wi h LMW and HMW chi osan. 365
3.4. Ba ie and mechanical p ope ies 366
Ligh ba ie p ope ies o chi osan/collagen ilms we e analyzed and UV- is 367
spec a a e shown in Figu e 4. As can be seen, collagen ilms p o ided a 368
maximum UV ligh ba ie om 200 o 250 nm, associa ed wi h ca bonyl, 369
ca boxyl, and amide g oups in he polypep ide chains o collagen (Pal, 370
Nidheesh, & Su esh, 2015; Vee u aj, A umugam, & Balasub amanian, 2013), 371
and a small abso p ion peak a 250-280 nm, associa ed o y osine and 372
phenylalanine amino acid esidues in collagen (Duan, Zhang, Du, Yao, & 373
Konno, 2009; Huang, Shiau, Chen, & Huang, 2011). The addi ion o chi osan 374
showed a s ong UV blocking capaci y, inc easing he abso bance a 250-280 375
nm due o he hyd oxyl auxoch ome g oups o chi osan (U anga e al., 2019). 376
200 300 400 500 600 700 800
Abso bance
Wa eleng h (nm)
Con ol
LMW
HMW
377
Figu e 4. UV- is spec a o chi osan/collagen ilms. 378
Bioma e ials o wound d essing need o main ain mois u e loss om he wound 379
a an op imum a e and, hus, a o e apo a ion and inhibi excess luid 380
19
abso p ion (Singh, Gup a, & Gup a, 2018). Taking he abo e in o conside a ion, 381
wa e apo ansmission a e was measu ed. Fo no mal skin, he inhe en 382
wa e apo e apo a ion a e is abou 204 g∙m-2∙day-1, while he e apo a ion 383
a e inc eases o 279 g∙m-2∙day-1 and 5138 g∙m-2∙day-1 o inju ed skin and bu n 384
skin, espec i ely (Lamke, Nilsson, & Rei hne , 1977). As can be seen in Table 385
3, he e was no signi ican (P > 0.05) di e ence in WVTR alues wi h he 386
addi ion o chi osan and all samples showed a high occlusi e cha ac e , 387
app op ia e o biomedical applica ions such as wound healing. Addi ionally, 388
WCA alues we e measu ed o de e mine he ilm hyd ophilic cha ac e , which 389
is known o ha e a g ea e ec on he in e ac ion be ween he bioma e ial and 390
he issue. As shown in Table 3, WCA alues signi ican ly (P < 0.05) dec eased 391
wi h he inco po a ion o chi osan, which would a o he adhesion o ib oblas s 392
and endo helial cells du ing wound healing. This dec ease o WCA alues has 393
also been epo ed o collagen- ucoidan ilms (Pe umal e al., 2018). 394
Table 3. Wa e apo ansmission a e (WVTR), wa e con ac angle (WCA), 395
ensile s eng h (TS), and elonga ion a b eak (EB) o d y and we 396
chi osan/collagen ilms. 397
Film WVTR
(g∙m-2∙day-1)
WCA
(º)
TSd y
(MPa)
EABd y
(%)
TSwe
(MPa)
EABwe
(%)
Con ol 1172a 103 ± 7c 7.7 ± 0.6a 13.2 ± 0.5a 2.0 ± 0.1a 50.6 ± 1.9a
LMW 1176a 90 ± 7a 12.4 ± 0.8b 24.1 ± 0.8b 2.8 ± 0.1b 34.0 ± 0.4b
HMW 1174a 93 ± 7b 13.0 ± 0.6b 21.9 ± 0.7c 2.3 ± 0.1c 39.3 ± 1.1c
a-cTwo means ollowed by he same le e in he same column a e no 398
signi ican ly (P > 0.05) di e en h ough he Tu key’s mul iple ange es . N = 5 399
was he minimum numbe o eplica ions. 400
Mechanical p ope ies o ilms a e la gely associa ed wi h dis ibu ion and 401
densi y o in e molecula and in amolecula in e ac ions in he ne wo k, so he 402
e ec o chi osan on mechanical p ope ies is shown in Table 3. Fo he d y 403
20
ilms, TS and EAB signi ican ly (P < 0.05) inc eased when chi osan was added,
404
induced by he in e ac ions be ween collagen and chi osan, as ound by FTIR
405
analysis. As p e iously shown in Table 2, collagen is mainly composed o α-
406
helix chains wi h in ense hyd ogen bonds and Van de Waals o ces among
407
hese chains. Due o he iple helix s uc u e, collagen shows app op ia e
408
ensile s eng h, bu he in ense in e ac ions among chains can p e en
409
s e ching. Howe e , in he we s a e, wa e can weaken hese in e ac ions
410
among collagen chains, acili a ing s e ching and p o iding he ilms wi h a
411
g ea e lexibili y. Ne e heless, his e ec was less no iceable o he ilms wi h
412
chi osan due o he addi ional in e ac ions among collagen and chi osan,
413
especially o he ilms wi h LMW chi osan, since he in e ac ions wi h smalle
414
size-molecules a e a o ed. This ac is in acco dance wi h he swelling
415
beha io shown in Figu e 1, whe e wa e up ake was highe o con ol ilms,
416
ollowed by he ilms wi h HMW chi osan and inally, by he ilms wi h LMW
417
chi osan. I is wo h no ing ha all ilms showed a ib il s uc u e in he ac u e
418
su ace a e mechanical es ing, as shown in Figu e 5.
419
420
21
Figu e 5. SEM images o he ac u e su ace in he b eak zone a e 421
mechanical analysis o a) con ol ilms, b) collagen ilms wi h LMW chi osan, 422
and c) collagen ilms wi h HMW chi osan. 423
3.5 Biodeg ada ion and cy o oxici y assessmen 424
On he one hand, deg ada ion s udies showed ha he e was no p og essi e 425
weigh dec ease due o he hyd oly ic ac ion a e 21 days in any o he 426
chi osan/collagen ilms. The ini ial loss, app oxima ely 15% o he ilms wi h 427
chi osan and 23% o he con ol ilms (Figu e 6A), was likely due o he 428
dissolu ion o glyce ol. These indings also sugges ed ha chi osan dec eased 429
he dissol ed glyce ol since he ilms wi h chi osan showed a lowe pe cen age 430
o deg aded weigh h ough he en i e assay and non-signi ican di e ences 431
we e obse ed in any ime-poin be ween he ilms wi h LMW o HMW chi osan. 432
The insoluble cha ac e o collagen in wa e explains he obse ed ilm 433
esis ance o hyd olysis. This p ope y was imp o ed wi h he addi ion o 434
chi osan ha o med hyd ogen bonds wi h collagen molecules, as p e iously 435
shown by FTIR analysis. 436
On he o he hand, enzyma ic deg ada ion s udies showed a comple e 437
deg ada ion o he con ol ilms in less han 12 h; howe e , o collagen ilms 438
wi h chi osan, only 27-28% o he weigh was los a 12 h (Figu e 6B). These 439
alues a e in acco dance wi h hose ound by Pe umal e al. (2018), who 440
epo ed 27% o deg ada ion a e 12 h o collagen ilms wi h 40% o ucoidan. 441
A e 96 h, app oxima ely 70% loss o he ini ial weigh was eached, wi h no 442
signi ican di e ences be ween he ilms wi h LMW and HMW chi osan. The 443
30% o he emaining weigh in he ilms wi h chi osan co esponded o 444
chi osan, which was no a ec ed by he enzyma ic ac ion. Thus, i is 445
22
demons a ed ha chi osan inc eased he esis ance o he collagen o he 446
ac ion o collagenase. These indings may be explained due o he ac ha he 447
enzyma ic deg ada ion depends on he numbe o clea age si es in he o ming 448
polyme and on he concen a ion o a ailable enzymes in he sca old 449
en i onmen (D u y & Mooney, 2003). In ha sense, he addi ion o chi osan 450
hinde ed he access o he enzyme o he clea age si es o collagen, educing 451
he deg ada ion caused by he collagenase. As p e iously epo ed (E xabide e 452
al., 2017), ilms o hese cha ac e is ics can be loaded wi h g ow h ac o s such 453
as epide mal g ow h ac o (EGF). The slow deg ada ion p o ile allows a 454
con olled elease o bioac i es, hus p o ec ing hem om he ha sh 455
mic oen i onmen wi h inc eased me allop o ease ac i i y p esen in 456
in lamma o y diseases such as ch onic wounds (Ba ien os, B em, S ojadino ic, 457
& Tomic-Canic, 2014). 458
459
Figu e 6. Deg ada ion and cy o oxici y assessmen : (A) hyd oly ic deg ada ion 460
wi h *** indica ing P < 0.001 o con ol s bo h LMW and HMW g oups and N.S. 461
indica ing non-signi ican di e ences be ween any ime-poin and day 1 o each 462
g oup; (B) enzyma ic deg ada ion wi h *** indica ing P < 0.001 o con ol s 463
bo h LMW and HMW g oups, # indica ing P < 0.05, and ### indica ing P < 0.001 464
be ween any ime-poin and 2 h o each g oup; (C) cy o oxici y assay wi h N.S. 465
indica ing non-signi ican di e ences s blank g oup and dashed line ma king 466
he 70% o cell iabili y. 467
23
Since ilms o biomedical applica ions mus ul ill addi ional equi emen s such 468
as biocompa ibili y, in i o cy o oxici y assay was pe o med ollowing an 469
adap ed p o ocol om he ISO 10993-5:2009 guidelines o biological e alua ion 470
o biomedical de ices. As can be seen in Figu e 6C, all he ilms showed mo e 471
han 70% o cell iabili y a e 48 h o di ec con ac wi h he ilm. These esul s 472
a e in ag eemen wi h he biocompa ibili y o each componen o he ilm o ming 473
o mula ion. Taking hese indings in o accoun , chi osan/collagen ilms a e in 474
compliance wi h he ISO 10993-5:2009 and may be conside ed as non-cy o oxic 475
bioma e ials. 476
Conclusions 477
Chi osan/collagen ilms wi h enhanced unc ional p ope ies o biomedical 478
applica ions such as wound healing we e de eloped. The addi ion o chi osan 479
educed he swelling deg ee o collagen ilms, which could allow a con olled 480
elease o epide mal g ow h ac o s, subs ances o in e es i ilms we e used as 481
wound d essings. This beha io was explained by he in e ac ions be ween 482
collagen and chi osan, as ound by FTIR analysis, which showed he 483
p e alence o he iple helix s uc u e o na i e collagen, as con i med by XRD 484
esul s. No signi ican di e ences we e ound in mos o unc ional p ope ies as 485
a unc ion o he molecula weigh o chi osan, including mechanical esis ance 486
in we s a e and a cell iabili y highe han 70% o all he ilms assessed. 487
Acknowledgmen s 488
Au ho s hank he Basque Go e nmen (KK-2019/00006) o inancial suppo . 489
M. Andonegi (PRE_2017_1_0025) and K. Las He as (PRE_2018_1_0412) 490
hank he Basque Go e nmen o hei PhD g an s. Thanks also Ad anced 491
Resea ch Facili ies (SGIke ) om he UPV/EHU. Au ho s also wish o hank he 492
24
in ellec ual and echnical assis ance om he ICTS “NANBIOSIS”, mo e 493
speci ically by he D ug Fo mula ion Uni (U10) o he CIBER-BBN a he 494
UPV/EHU. 495
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