Academic Edi o : Iolanda De Ma co
Recei ed: 19 No embe 2024
Re ised: 2 Janua y 2025
Accep ed: 7 Janua y 2025
Published: 8 Janua y 2025
Ci a ion: G anados-Ca e a, C.M.;
Cas o-C iado, D.; Abdullah, J.A.A.;
Jiménez-Rosado, M.; Pe ez-Puyana,
V.M. Ae ogels Based on Chi osan and
Collagen Modi ied wi h Fe2O3and
Fe3O4Nanopa icles: Fab ica ion and
Cha ac e iza ion. Polyme s 2025,17,
133. h ps://doi.o g/10.3390/
polym17020133
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
Ae ogels Based on Chi osan and Collagen Modi ied wi h Fe2O3
and Fe3O4Nanopa icles: Fab ica ion and Cha ac e iza ion
Ca men Mª G anados-Ca e a 1, Daniel Cas o-C iado 1, Joha Amin Ahmed Abdullah 1,
Me cedes Jiménez-Rosado 2,* and Víc o M. Pe ez-Puyana 3,*
1Depa men o Chemical Enginee ing, Facul y o Chemis y, Uni e si y o Se ille, 41012 Se ille, Spain;
[email p o ec ed] (C.M.G.-C.); [email p o ec ed] (D.C.-C.); [email p o ec ed] (J.A.A.A.)
2Depa men o Applied Chemis y and Physics, Facul y o Biological and Ambien al Sciences,
Uni e si y o León, 24009 León, Spain
3Depa men o Enginee ing and Ma e ials Science and T anspo a ion, Uni e si y o Se ille,
41092 Se ille, Spain
*Co espondence: [email p o ec ed] (M.J.-R.); [email p o ec ed] (V.M.P.-P.)
Abs ac : The necessi y o mi iga e he in insic issues associa ed wi h issue o o gan
ansplan s, in o de o add ess he ising p e alence o diseases a ibu able o inc eased
li e expec ancy, p o ides a a ionale o he pu sui o inno a ion in he ield o bioma e ials.
Speci ically, biopolyme ic ae ogels ep esen a signi ican ad ancemen in he ield o issue
enginee ing, o e ing a p omising solu ion o he o ma ion o empo a y po ous ma ices
ha can eplace damaged issues. Howe e , he unc ional cha ac e is ics o hese ma e ials
a e inadequa e, necessi a ing he implemen a ion o ma ix ein o cemen me hods o
enhance hei pe o mance. In his s udy, chemical and g een i on oxide nanopa icles,
p e iously syn hesized and documen ed in exis ing esea ch, we e inco po a ed in o hyb id
ae ogels combining collagen (C) and chi osan (CH). The cha ac e iza ion o hese ae ogels
was conduc ed h ough heological, mic os uc u al, and unc ional analyses. The esul s
demons a e ha he inco po a ion o i on oxide nanopa icles has a signi ican in luence
on he p ope ies o he ae ogels ab ica ed wi h hem. In pa icula , he inco po a ion
o hese nanopa icles has been obse ed o modi y he mechanical p ope ies, wi h an
inc ease in s eng h and po osi y ha may suppo cell p oli e a ion.
Keywo ds: chi osan; collagen; ae ogels; issue enginee ing; nanopa icles; i on oxide;
bioma e ials
1. In oduc ion
The exponen ial g ow h in he numbe o indi iduals wi h one o mo e ch onic
diseases as a consequence o he inc ease in global li e expec ancy is esul ing in a co -
esponding ise in he numbe o o gan ansplan s equi ed. Howe e , he demand o
hese ansplan s canno be me due o he insu icien quan i y o a ailable o gans [
1
,
2
].
Consequen ly, scien is s a e concen a ing hei e o s on he c ea ion o inno a i e bioma-
e ials ha can egene a e, eplace, and epai issues, wi h he aim o educing he isk o
in ec ions associa ed wi h con en ional ea men s [
3
,
4
]. In his con ex , bioma e ials a e a
di e se ange o ma e ials, including me al, ce amic, and polyme ic compounds, which
a e designed o be biocompa ible ( o a oid ad e se eac ions when in con ac wi h hos
issue), bioac i e ( o s imula e issue esponses), biodeg adable ( o disappea a e he issue
is healed), and s e ilizable, allowing hei use in issue enginee ing o he eplacemen
o damaged s uc u es [
5
–
8
]. In pa icula , he main objec i e o issue enginee ing is
Polyme s 2025,17, 133 h ps://doi.o g/10.3390/polym17020133
Polyme s 2025,17, 133 2 o 15
he de elopmen o sca olds ha mimic he dynamics o he ex acellula ma ix (ECM)
and egula e cell adhesion, mig a ion, and o he p ocesses [
9
,
10
]. Howe e , his is no as
simple as i seems since he e a e issues ha equi e speci ic equi emen s a e ansplan-
a ion (such as hea al es o blood essels, o example); he e o e, knowledge o he
mechanical– unc ional p ope ies o hese issues will be necessa y [11–13].
Bioma e ials can be syn hesized by a numbe o di e en ab ica ion me hods, includ-
ing con en ional echniques (e.g., cas ing, phase sepa a ion and lyophiliza ion, syn he i-
za ion, au o-assembly, o elec ospinning) o addi i e echniques (e.g., s e eoli hog aphy,
used-deposi ion modeling (FDM), o h ee-dimensional p in ing) [
8
,
14
,
15
]. Howe e ,
among he di e en bioma e ials, ae ogels s and ou due o hei dis inc i e physical s uc-
u e, comp ising h ee-dimensional po ous solid ne wo ks wi h an excep ionally high
speci ic su ace a ea [
16
]. Speci ically, his ype o ma e ial can be elabo a ed by he sub-
s i u ion o he liquid inside a gel wi hou any signi ican change in hei s uc u e. They
also exhibi o he ad an ages, including low densi y, unable chemical p ope ies, he mal
esis ance, and high loading capaci y, which makes hem app op ia e o a wide ange
o applica ions, including wa e ea men , he mal insula ion, he ood indus y, and
medical and pha maceu ical applica ions, among o he s [
17
–
19
]. Consequen ly, ae ogels
a e a ac ing inc easing in e es as a means o deli e ing a ange o ac i e compounds o
he apeu ic d ugs, among o he applica ions [
20
]. The e a e se e al me hods ha can be
employed o he ab ica ion o hese sca olds, such as 3D biop in ing, elec ospinning, o
he use o hyd ogel [
21
–
24
]. The la e me hod, commonly e e ed o as phase sepa a ion,
has gained signi ican ac ion due o i s capaci y o abso b subs an ial quan i ies o wa e .
I ypically necessi a es a eeze-d ying p ocess o sublima e he sol en , ul ima ely leading
o he o ma ion o po ous s uc u es [23].
Speci ically, he o ma ion o ae ogels in ol es he use o a a ie y o aw ma e ials, such
as allo opic ca bon, me al oxides, o polyme s, which can be syn he ic o na u al. Ne e he-
less, he use o na u al polyme s, also designa ed as biopolyme s, which a e enewable and
plen i ul, is cu en ly expe iencing a su ge in in e es due o hei a o able p ope ies, includ-
ing exempla y cell a achmen , biodeg adabili y, and biocompa ibili y [
2
,
18
]. Biopolyme s
can be classi ied in o wo main ca ego ies: polysaccha ides and p o eins. Polysaccha ides
include subs ances such as algina e, s a ch, cellulose, chi in, chi osan, and aga . P o-
eins, on he o he hand, encompass subs ances like gela in, collagen, and soy [
2
,
25
,
26
].
These polyme s acili a e he o ma ion o bo h co alen and non-co alen chemical bonds,
he eby o e coming he limi a ions o o he ma e ials in e ms o cell adhesion and cellula
a achmen [7].
Collagen (CG) is a p o ein ha plays a pi o al ole in he main enance o he ECM. I
is one o he mos abundan p o eins in his ma ix, comp ising h ee polypep ide chains
ha a e linked oge he o o m a iple-helix s uc u e [
27
,
28
]. In pa icula , he e a e up o
29 ypes o collagens, al hough he mos abundan is ype I collagen [
3
]. This is ound in
bones, ee h, skin, and ligamen s. This ib ous p o ein is cons i u ed by p oline, glycine,
and hyd oxyp oline [
3
,
28
] and has been widely used in ecen yea s o he de elopmen
o no el sca olds which combine excellen biocompa ibili y, low oxici y, and os eogenic
induc ion p ope ies [
29
–
33
]. Conside ing p e ious s udies, collagen is a biopolyme ha
has been used o he o ma ion o ae ogel ha suppo s bone egene a ion as a esul o i s
abili y o bone biomine aliza ion ac i i y as well as high a e o cell p oli e a ion [
34
] o
o ca ilage issue epai due o i s geome y, which is simila o he one ha is possessed
by he biopolyme , among o he s [35,36].
On he o he hand, chi osan (CH) is a na u al polyme de i ed om he deace yla ion
and enzyma ic deg ada ion o chi in, which allows o he u iliza ion o ood was e (espe-
cially shells om c us aceans and insec s). This polysaccha ide is no able o he p esence o
Polyme s 2025,17, 133 3 o 15
amino g oups in i s chemical s uc u e and o i s abundance and en i onmen ally iendly
na u e [
37
–
39
]. Mo eo e , he u iliza ion o his polysaccha ide has been demons a ed o
p omo e addi ional p ope ies, such as an i i al, an ibac e ial, o an i ungal cha ac e is-
ics, which a e essen ial o issue enginee ing [
40
]. This has been e idenced by p e ious
s udies [
41
–
43
]. Fo ins ance, he inco po a ion o chi osan allowed he c ea ion o di e en
ype o sca olds, such as empe a u e-sensi i e sys ems, whose applica ion can be ound
in se e al a eas, such as neu odegene a i e diseases, join diseases, and den is y, among
o he s [44–46].
In any case, he combina ion o hese biopolyme s (CG and CH) p esen s se e al
disad an ages, including poo mechanical p ope ies [
29
,
32
,
40
]. Consequen ly, esea che s
a e a emp ing o de elop no el al e na i es, such as polyme blending [
40
,
47
] o he
inco po a ion o nanopa icles (NPs) [
36
,
48
], wi h he objec i e o a aining he desi ed
p ope ies. In his con ex , nanoma e ials a e e ined as a wide ange o ma e ials whose
dimensions a e wi hin he ange o 1–100 nm. They a e ob ained om he manipula ion
o ma e ials h ough chemical o physical p ocesses [
49
,
50
]. The p ope ies o his ype
o ma e ial a e dependen on i s size due o he la ge numbe o supe icial a oms in
compa ison o i s olume, which esul s in a mo e ac i e su ace [
51
,
52
]. The syn hesis o
hese ma e ials can be achie ed h ough a a ie y o me hods, depending on he desi ed
p ope ies and in ended applica ions. These me hods can be b oadly classi ied as ei he
op-down (e.g., mechanical g inding and lase abla ion) [
53
,
54
] o bo om-up echniques
(e.g., elec ic a c, lame syn hesis, apo phase deposi ion, mic oemulsion, sol–gel, colloidal
syn hesis, and biological syn hesis) [
55
–
60
]. Thus, Rahman e al. de eloped ae ogels
ein o ced wi h i on oxide NPs h ough eeze-d ying, esul ing in ma e ials wi h a high
speci ic su ace a ea and s abili y in wa e [
61
]. Ne e heless, he in eg a ion o hese NPs
is pa icula ly p omising in he biomedical ield, whe e hey can acili a e he con olled
elease o d ugs, enhancing hei he apeu ic e icacy and biological, mechanical, and
elec ical p ope ies [
62
]. Howe e , among all he nanopa icles, magne i e is one o he
mos equen ly used e i es due o i s non oxici y, allowing he o ma ion o d ug deli e y
sys ems as a esul o he inc ease in he a ini y and a ge abili y o cells [
63
]. Speci ically,
he addi ion o magne i e in o gel ma ix p omo es he o ma ion o a highly po ous gel [
64
],
as eco ded in p e ious s udies whe e his compound was added in o a sol–gel solu ion,
imp o ing blood abso p ion and mild cy o oxic e ec s [
36
], o he de elopmen o sca olds
o ca diac issue enginee ing [
65
] o o he o ma ion o hyb id biopolyme -based ae ogels,
which p omo es he adso p ion o hea y me al ions and o ms a high po ous in e connec ed
ma ix [66].
The e o e, he objec i e o his wo k was he de elopmen o a p elimina y s udy o
he ab ica ion o di e en ae ogels modi ied wi h nanopa icles. Thus, hyb id ae ogels
based on collagen and chi osan we e c ea ed using he phase sepa a ion me hod, ein o cing
hem wi h i on oxide nanopa icles ob ained h ough wo di e en me hods (chemical
o g een syn hesis). The physicochemical, mechanical, and unc ional p ope ies o hese
ae ogels we e e alua ed in o de o compa e he di e en sys ems. Thus, he main no el y
o his a icle lies in he combina ion o nanopa icles o he o ma ion o collagen–chi osan-
based hyb id bioma e ials and he compa ison o he p ope ies obse ed in he modi ied
ma e ials wi h di e en i on oxide nanopa icles.
2. Ma e ials and Me hods
2.1. Ma e ials
The biopolyme s used o he p epa a ion o he ae ogels we e ype I collagen om
pigs (HI95), wi h a p o ein con en highe han 95 w %, supplied by Essen ia P o ein
Solu ions S.A. (G ås en, Denma k) and low-molecula -weigh chi osan (molecula weigh
Polyme s 2025,17, 133 4 o 15
(MW) o 130,000 g/mol and wi h a deace yla ion deg ee o 75–85%), supplied by Sigma
Ald ich (San Luis, MO, USA). The collagen was selec ed due o i s high p o ein con en ,
while he chi osan was chosen o i s abili y o o m s able gels a low concen a ions.
Fu he mo e, 0.05 M ace ic acid a pH 3.2 was used as a sol en o acili a e he solubiliza ion
o he biopolyme s.
Addi ionally, chemically syn hesized (Ch-NPs) and en i onmen ally iendly syn-
he ized (G -NPs) magne ic i on oxide nanopa icles (mix u e o Fe
2
O
3
and Fe
3
O
4
) we e
included in his s udy, sou ced om p e iously published wo ks [
67
,
68
]. The G -NPs
con ained 94.6% Fe
3
O
4
and 3.4% Fe
2
O
3
, wi h an a e age diame e o 6.6
±
4.9 nm.
The Ch-NPs we e composed o 86.5% Fe
3
O
4
and 13.3% Fe
2
O
3
, wi h an a e age diam-
e e o
16.8 ±1.4 nm.
These nanopa icles exhibi magne ic p ope ies and s abili y ha
make hem sui able o he enhancemen o he s uc u al and unc ional p ope ies o
biopolyme ic ae ogels.
2.2. Ae ogel P ocessing
The ae ogels we e ob ained h ough a p ocessing me hod based on he me hodology
p oposed by Pe ez-Puyana e al. [
69
], as shown in Figu e 1. Thus, an ae ogel wi hou
nanopa icles (AG-Re ) was p oduced, comp ising a o al biopolyme con en o 2 w/ %
(1 w/ %
o CG and 1 w/ % o CH). The biopolyme s, oge he wi h he 20 mL o solu ion,
we e placed in a Sigma 3–18k cen i uge (MEDIFRIGER BL-S, J.P Selec a, Ba celona, Spain)
and subjec ed o cen i uga ion a 10,000 pm and 4
◦
C o a pe iod o 7 min. Subsequen ly,
he samples we e ozen a
−
40
◦
C o 1.5–2 h and subsequen ly placed in a eeze-d ye
(LyoQues , TELSTAR, Ba celona, Spain) o 24 h a 0.01 mba and
−
80
◦
C o sublima e
he sol en .
Polyme s 2025, 17, x FOR PEER REVIEW 4 o 16
2. Ma e ials and Me hods
2.1. Ma e ials
The biopolyme s used o he p epa a ion o he ae ogels we e ype I collagen om
pigs (HI95), wi h a p o ein con en highe han 95 w %, supplied by Essen ia P o ein So-
lu ions S.A. (G ås en, Denma k) and low-molecula -weigh chi osan (molecula weigh
(MW) o 130,000 g/mol and wi h a deace yla ion deg ee o 75–85%), supplied by Sigma
Ald ich (San Luis, MO, USA). The collagen was selec ed due o i s high p o ein con en ,
while he chi osan was chosen o i s abili y o o m s able gels a low concen a ions. Fu -
he mo e, 0.05 M ace ic acid a pH 3.2 was used as a sol en o acili a e he solubiliza ion
o he biopolyme s.
Addi ionally, chemically syn hesized (Ch-NPs) and en i onmen ally iendly syn-
he ized (G -NPs) magne ic i on oxide nanopa icles (mix u e o Fe2O3 and Fe3O4) we e
included in his s udy, sou ced om p e iously published wo ks [67,68]. The G -NPs con-
ained 94.6% Fe3O4 and 3.4% Fe2O3, wi h an a e age diame e o 6.6 ± 4.9 nm. The Ch-NPs
we e composed o 86.5% Fe3O4 and 13.3% Fe2O3, wi h an a e age diame e o 16.8 ± 1.4
nm. These nanopa icles exhibi magne ic p ope ies and s abili y ha make hem sui able
o he enhancemen o he s uc u al and unc ional p ope ies o biopolyme ic ae ogels.
2.2. Ae ogel P ocessing
The ae ogels we e ob ained h ough a p ocessing me hod based on he me hodology
p oposed by Pe ez-Puyana e al. [69], as shown in Figu e 1. Thus, an ae ogel wi hou na-
nopa icles (AG-Re ) was p oduced, comp ising a o al biopolyme con en o 2 w/ % (1
w/ % o CG and 1 w/ % o CH). The biopolyme s, oge he wi h he 20 mL o solu ion,
we e placed in a Sigma 3–18k cen i uge (MEDIFRIGER BL-S, J.P Selec a, Ba celona,
Spain) and subjec ed o cen i uga ion a 10,000 pm and 4 °C o a pe iod o 7 min. Sub-
sequen ly, he samples we e ozen a −40 °C o 1.5–2 h and subsequen ly placed in a
eeze-d ye (LyoQues , TELSTAR, Ba celona, Spain) o 24 h a 0.01 mba and −80 °C o
sublima e he sol en .
Figu e 1. Syn hesis o collagen–chi osan-based ae ogel ( e e ence sys em and sys ems wi h nano-
pa icles inco po a ed).
Figu e 1. Syn hesis o collagen–chi osan-based ae ogel ( e e ence sys em and sys ems wi h nanopa -
icles inco po a ed).
The a io o he base mix u e emained unchanged o he ae ogels wi h inco po a ed
nanopa icles. The e o e, he same quan i ies o collagen and chi osan we e used. The
main di e ence in his case is ha only 15 mL o 0.05 M ace ic acid oge he wi h he
weighed biopolyme s we e cen i uged. The emaining 5 mL was combined wi h 8 mg o
nanopa icles (2 w/ % wi h espec he o al biopolyme con en ) in a es ube. This es
Polyme s 2025,17, 133 5 o 15
ube was placed in an ul asound machine (J.P. Selec a S.A., Spain) o 15 min o acili a e
he dispe sion o he nanopa icles in he sol en . Bo h samples (biopolyme solu ion a e
cen i uga ion and nanopa icle suspension) we e mixed be o e he eezing s ep.
2.3. Ae ogel Cha ac e iza ion
2.3.1. Mechanical P ope ies
Dynamic comp ession es s we e conduc ed o he pu pose o cha ac e izing he me-
chanical p ope ies o he ae ogels. Fo his pu pose, an RSA3 heome e (TA Ins umen s,
New Cas le, DE, USA) wi h a pla e–pla e geome y (dia. o 15 mm) was used. Fi s ly,
s ain sweep es s we e ca ied ou a 1 Hz wi hin a s ain ange o 2.5
·
10
−4
% o 2.5%
o de e mine he linea iscoelas ic ange and he c i ical s ain (las s ain in he linea
iscoelas ic ange) o he ae ogels. Subsequen ly, equency sweep es s we e pe o med
be ween 0.02 and 20 Hz a a cons an s ain wi hin he linea iscoelas ic ange, wi h he
objec i e o ob aining he alues o he elas ic modulus (E
′
), he iscous modulus (E
′′
), and
he loss angen ( an δ= E′′/E′) as a unc ion o equency.
2.3.2. Op ical P ope ies: Colo Measu emen s
A colo ime y es was pe o med o ca y ou a colo analysis o he samples ab ica ed,
using a high-p ecision colo ime e MERHOVO (model NR110). Thus, he alues o L* (i.e.,
ligh ness) and a* and b* ( ed, g een, blue, and yellow in CIELAB colo space) we e ound
o each sample as he a e age o i e scans. In pa icula , b* d i s om nega i e alues
(blue) o posi i e alues (yellow), whe eas a* d i s om nega i e alues o posi i e alues
associa ed wi h g een and ed, espec i ely.
2.3.3. Scanning Elec on Mic oscopy (SEM)
To analyze he mic os uc u e o he ae ogels, a scanning elec on mic oscope, Zeiss
EVO (Zeiss, Obe kochen, Ge many), wi h a seconda y elec on de ec o was used wi h an
accele a ing ol age o 10 kV. The samples we e p e iously coa ed wi h a laye (less han
10 nm
hick) o Pd/Au and ea ed wi h 1% osmium apo o 8 h o ix he sca old s uc u e
and imp o e he quali y o he ob ained mic og aphs. These images we e analyzed using
ImageJ so wa e, e sion 1.54g (Na ional Ins i u e o Heal h, Be hesda, MD, USA).
On he o he hand, he o al po osi y o he ae ogels was calcula ed based on he
me hod o indi ec measu emen o he ee olume inside he sca old [
70
]. The ou ypes
o ae ogels we e measu ed and weighed o calcula e hei densi ies (
ρsca old
). The o al
po osi y was ob ained using Equa ion (1):
ε(%)=1−ρsca old
ρma e ial ×100 (1)
whe e ρma e ial is he a e age densi y o chi osan and collagen.
2.3.4. Func ional Cha ac e iza ion
To e alua e he an ioxidan ac i i y o he ae ogels, a p e ious p o ocol was ol-
lowed [
67
,
68
], which was o iginally applied o he nanopa icles. B ie ly, 4 mg o each
ae ogel sample was dissol ed in a mix u e con aining 1 mL o DMSO and 1 mL o DPPH so-
lu ion. Then, he samples we e s i ed igo ously o 30 s and subsequen ly le in da kness
o 30 min. Finally, he abso bance was measu ed a 517 nm in a spec opho ome e U-1100
(Hi achi, Chiyoda, Japan). The inhibi ion pe cen age was calcula ed using Equa ion (2):
Inhibi ion(%) = Abso bance o con ol −Abso bance o sample
Abso bance o con ol ×100 (2)
Polyme s 2025,17, 133 6 o 15
This equa ion allows o he quan i ica ion o an ioxidan ac i i y by compa ing he
abso bance o he con ol solu ion wi h ha o he ae ogel samples.
The he mal s abili y o he ae ogels was s udied a 50
◦
C and 100% ela i e humidi y.
Fo his pu pose, one-qua e o each sca old was cu and placed on Pe i dishes. They
we e hen placed in a double-bo omed con aine . The lowe pa was illed wi h a solu ion
o sal in wa e , achie ing a humidi y o 100%, while he uppe pa was illed wi h he ou
samples. Finally, he con aine was placed in an o en a 50
◦
C. Pho os we e aken a 0, 1, 2,
12, 24, 48, and 60 h o obse e he deg ada ion p ocess o he ae ogels o e ime.
2.4. S a is ical Analysis
A leas h ee eplica es o each sample we e ob ained in o de o e alua e he eplica-
bili y and ep oducibili y o he esul s. Signi ican di e ences we e e alua ed wi h - es s
a a con idence le el o 95% (p< 0.05).
3. Resul s and Discussion
3.1. Ae ogel Cha ac e iza ion
3.1.1. Mac os uc u al Appea ance o he Ae ogels
Figu e 2shows he mac os uc u al appea ance o he elabo a e ae ogels. As can be
seen, he e e ence sys em (AG-Re , Figu e 2A) exhibi s a whi e colo a ion in acco dance
wi h i s composi ion. Ne e heless, he inco po a ion o nanopa icles esul s in a change in
colo a ion o o ange. Thus, he inco po a ion o chemical nanopa icles (Ch-NPs, Figu e 2B)
o g een nanopa icles (G -NPs, Figu e 2C) esul s in he o ma ion o an o ange hue
in he sys ems, wi h he Ch-NPs sys ems exhibi ing a da ke o ange colo a ion. This
da kening may be a ibu ed o he la ge size o he Ch-NPs, which allows o g ea e colo
p ominence wi hin he s uc u e.
Polyme s 2025, 17, x FOR PEER REVIEW 6 o 16
30 min. Finally, he abso bance was measu ed a 517 nm in a spec opho ome e U-1100
(Hi achi, Chiyoda, Japan). The inhibi ion pe cen age was calcula ed using Equa ion (2):
Inhibi ion (%)=(Abso bance o con ol − Abso bance o sample
Abso bance o con ol )×100
(2)
This equa ion allows o he quan i ica ion o an ioxidan ac i i y by compa ing he
abso bance o he con ol solu ion wi h ha o he ae ogel samples.
The he mal s abili y o he ae ogels was s udied a 50 °C and 100% ela i e humidi y.
Fo his pu pose, one-qua e o each sca old was cu and placed on Pe i dishes. They
we e hen placed in a double-bo omed con aine . The lowe pa was illed wi h a solu-
ion o sal in wa e , achie ing a humidi y o 100%, while he uppe pa was illed wi h
he ou samples. Finally, he con aine was placed in an o en a 50 °C. Pho os we e aken
a 0, 1, 2, 12, 24, 48, and 60 h o obse e he deg ada ion p ocess o he ae ogels o e ime.
2.4. S a is ical Analysis
A leas h ee eplica es o each sample we e ob ained in o de o e alua e he epli-
cabili y and ep oducibili y o he esul s. Signi ican di e ences we e e alua ed wi h -
es s a a con idence le el o 95% (p < 0.05).
3. Resul s and Discussion
3.1. Ae ogel Cha ac e iza ion
3.1.1. Mac os uc u al Appea ance o he Ae ogels
Figu e 2 shows he mac os uc u al appea ance o he elabo a e ae ogels. As can be
seen, he e e ence sys em (AG-Re , Figu e 2A) exhibi s a whi e colo a ion in acco dance
wi h i s composi ion. Ne e heless, he inco po a ion o nanopa icles esul s in a change
in colo a ion o o ange. Thus, he inco po a ion o chemical nanopa icles (Ch-NPs, Figu e
2B) o g een nanopa icles (G -NPs, Figu e 2C) esul s in he o ma ion o an o ange hue
in he sys ems, wi h he Ch-NPs sys ems exhibi ing a da ke o ange colo a ion. This da k-
ening may be a ibu ed o he la ge size o he Ch-NPs, which allows o g ea e colo
p ominence wi hin he s uc u e.
Figu e 2. Mac os uc u al appea ance o he ae ogels elabo a ed. (A) Re e ence, (B) chemical nano-
pa icles inco po a ed, and (C) g een nanopa icles inco po a ed.
A
B
C
Figu e 2. Mac os uc u al appea ance o he ae ogels elabo a ed. (A) Re e ence, (B) chemical
nanopa icles inco po a ed, and (C) g een nanopa icles inco po a ed.
3.1.2. Op ical P ope ies: Colo Measu emen s
As shown p e iously, he e a e sligh di e ences be ween he mac os uc u al appea -
ance o he di e en samples. The alues associa ed wi h he colo pa ame e s a e shown in
Table 1. The e e ence sys em associa ed wi h a collagen–chi osan-based ae ogel p esen ed
Polyme s 2025,17, 133 7 o 15
a whi ish colo as shown in he alues o L*, a*, and b*. Rega ding he samples which
inco po a e nanopa icles, he e is a da kening o he samples, as well as an inc ease in a*
and b* alues as a esul o he addi ion o b own-o ange nanopa icles.
Table 1. Colo pa ame e s o each sys em: e e ence (Re ), chemical nanopa icles included (Ch), and
g een nanopa icles included (G ). Di e en le e s o symbols mean signi ican di e ences (p< 0.05).
Sample L*∆L b*∆b a*∆a
Re 83.9 ±6.8 a-2.5 ±0.5 A- 10.2 ±2.0 α-
Ch 70.7 ±2.4 b−13.2 18.3 ±4.0 B15.8 20.9 ±6.3 β10.8
G 77.2 ±6.6 b−6.7 12.2 ±1.0 C9.7 23.1 ±1.0 β13.0
3.1.3. Mechanical P ope ies
The esul s o he equency sweep es s o he di e en sys ems a e shown in Figu e 3.
I can be obse ed ha he Re and Ch sys ems exhibi modulus alues ( o bo h E
′
and
E
′′
) ha a e p ac ically s able a he en i e equency ange s udied. Ne e heless, he G
sys em p esen s a sligh a ia ion in hese alues, pa icula ly in E
′′
. This beha io could
be a ibu ed o he inhe en ly uns able na u e o he G -NPs, which is a consequence o he
p esence o polyphenols du ing he manu ac u ing p ocess. The polyphenols emain on
he su ace o he nanopa icles, he eby c ea ing a sys em ha is mo e uns able.
Polyme s 2025, 17, x FOR PEER REVIEW 7 o 16
3.1.2. Op ical P ope ies: Colo Measu emen s
As shown p e iously, he e a e sligh di e ences be ween he mac os uc u al ap-
pea ance o he di e en samples. The alues associa ed wi h he colo pa ame e s a e
shown in Table 1. The e e ence sys em associa ed wi h a collagen–chi osan-based ae ogel
p esen ed a whi ish colo as shown in he alues o L*, a*, and b*. Rega ding he samples
which inco po a e nanopa icles, he e is a da kening o he samples, as well as an inc ease
in a* and b* alues as a esul o he addi ion o b own-o ange nanopa icles.
Table 1. Colo pa ame e s o each sys em: e e ence (Re ), chemical nanopa icles included (Ch),
and g een nanopa icles included (G ). Di e en le e s o symbols mean signi ican di e ences (p <
0.05).
Sample
L*
ΔL
b*
Δb
a*
Δa
Re
83.9 ± 6.8 a
-
2.5 ± 0.5 A
-
10.2 ± 2.0 α
-
Ch
70.7 ± 2.4 b
−13.2
18.3 ± 4.0 B
15.8
20.9 ± 6.3 β
10.8
G
77.2 ± 6.6 b
−6.7
12.2 ± 1.0 C
9.7
23.1 ± 1.0 β
13.0
3.1.3. Mechanical P ope ies
The esul s o he equency sweep es s o he di e en sys ems a e shown in Figu e
3. I can be obse ed ha he Re and Ch sys ems exhibi modulus alues ( o bo h E′ and
E″) ha a e p ac ically s able a he en i e equency ange s udied. Ne e heless, he G
sys em p esen s a sligh a ia ion in hese alues, pa icula ly in E″. This beha io could
be a ibu ed o he inhe en ly uns able na u e o he G -NPs, which is a consequence o
he p esence o polyphenols du ing he manu ac u ing p ocess. The polyphenols emain
on he su ace o he nanopa icles, he eby c ea ing a sys em ha is mo e uns able.
Figu e 3. F equency sweep es s o he di e en ae ogels: e e ence (Re ), chemical nanopa icles
included (Ch), and g een nanopa icles included (G ).
Table 2 shows he alues o he elas ic modulus a 1 Hz (E′1), c i ical s ain (εc i ), and
loss angen a 1 Hz ( an δ1) in o de o acili a e he compa ison be ween he sys ems. All
sys ems exhibi solid cha ac e ( an δ1 < 1) wi hou signi ican di e ences. On he o he
hand, he elas ic modulus was ma kedly highe o he Re and Ch sys ems, and i was
Figu e 3. F equency sweep es s o he di e en ae ogels: e e ence (Re ), chemical nanopa icles
included (Ch), and g een nanopa icles included (G ).
Table 2shows he alues o he elas ic modulus a 1 Hz (E
′1
), c i ical s ain (
εc i
), and
loss angen a 1 Hz ( an
δ1
) in o de o acili a e he compa ison be ween he sys ems. All
sys ems exhibi solid cha ac e ( an
δ1
< 1) wi hou signi ican di e ences. On he o he
hand, he elas ic modulus was ma kedly highe o he Re and Ch sys ems, and i was
obse ed ha hese alues a e simila o hose ob ained o applica ions such as b ains
(15 kPa),
kidneys (50 kPa), panc eas, i is, o lungs (80 kPa) as shown in he li e a u e [
13
,
71
].
This di e en beha io is simila ly e iden in he c i ical s ain, whe ein he Re sys em
exhibi s he lowes alue and he G sys em he highes one. These esul s may be a ibu ed
Polyme s 2025,17, 133 8 o 15
o he p esence o polyphenols in he G sys ems, which ha e been demons a ed o gene a e
ionic o ces ha could impede he in e ac ion o NPs wi h he biopolyme s.
Table 2. Elas ic modulus and loss angen a 1 Hz (E
′1
and an
δ1
, espec i ely) and c i ical s ain (
εc i
)
alues o each sys em: e e ence (Re ), chemical nanopa icles included (Ch), and g een nanopa icles
included (G ). Di e en le e s o symbols mean signi ican di e ences (p< 0.05).
Sample E′1(kPa) an δ1(-) εc i (%)
Re 27.46 ±18.24 α0.10 ±0.05 a0.34 ±0.15 A
Ch 25.74 ±10.19 α0.13 ±0.01 a0.79 ±0.01 B
G 5.79 ±4.43 α0.15 ±0.02 a1.63 ±0.53 C
3.1.4. Scanning Elec on Mic oscopy (SEM)
The mic og aphs o he h ee ae ogels a e p esen ed in Figu e 4. They show an i egula
s uc u e, cha ac e ized by he p esence o mic o- and mac o-po es. The Re and Ch ae ogels
show e y simila lamina s uc u es (Figu e 4A,B), wi h a de ined di ec ionali y. This
may accoun o he minimal di e ence in modulus obse ed in he heological s udy.
The G ae ogel also exhibi s a lamina s uc u e, bu no di ec ional, and displays g ea e
he e ogenei y (Figu e 4C). The low alues o he elas ic and iscous moduli can be a ibu ed
o he lack o op imal s acking, as a esul o he exis ence o a highe concen a ion o
solids in he ma ix ha al e he mo phology o he ae ogels, p omo ing a minimiza ion
in he dimension o he po e opening as eco ded in p e ious wo ks [
72
,
73
]. In his sense,
di e en wo ks e eal ha his i egula shape is ypical o his ype o ma e ial, o ming
an in e connec ed po ous s uc u e ha is necessa y o enhancing cell mig a ion and
he anspo a ion o biomolecules [
74
–
76
]. This s uc u e, which is composed o he
supe posi ion o di e en laye s, may be linked o he gela ion p ocedu e ha is needed o
he o ma ion o he ae ogel by he lyophiliza ion o a hyd ogel [77].
Rega ding po osi y, while he Re and Ch ae ogels display a mul i ude o po es
exhibi ing he e ogeneous sizes and shapes (wi h a mean po osi y o 46.5
±
15.8 nm and
29.4
±
17.4 nm, espec i ely) and a ela i ely uni o m dispe sion. On he o he hand,
G ae ogels a e de oid o po es, wi h he exis ing ones being uni o mly minu e. This
sugges s ha he ae ogels s udied possess an adequa e po e size, which would acili a e cell
egene a ion and g ow h in hei po en ial applica ion as a bioma e ial. Consequen ly, o
acili a e a mo e p ecise compa ison o he po osi y o he ae ogels, he pe cen age o o al
po osi y o each ma e ial has been calcula ed and is p esen ed in Table 3. In gene al, all
ae ogels show adequa e po osi y alues (>98%), which a e g ea e han he necessa y 85%
o mos o he applica ions [
78
] and which can ac as a a o able poin o applica ions such
as bone and ca ilage egene a ion as shown in he li e a u e [
79
]. As can be obse ed in
he able abo e, he heo ical po osi y o AG-G is he highes , which does no co espond
o wha has been discussed o he SEM images. This is due o he ac ha he calcula ed
po osi y e e s o he o al olume o he ae ogel, while he SEM echnique allows us o
s udy only he laye o he ma e ial and po es ha can be ound inside he sca old.
Polyme s 2025,17, 133 9 o 15
Polyme s 2025, 17, x FOR PEER REVIEW 9 o 16
Figu e 4. Mic og aphs o di e en ae ogels made: (A) e e ence, (B) chemical nanopa icles in-
cluded, and (C) g een nanopa icles inco po a ed.
Table 3. Theo e ical po osi y alues o each ae ogel s udied: e e ence (Re ), chemical nanopa icles
included (Ch), and g een nanopa icles included (G ). Di e en le e s mean signi ican di e ences
(p < 0.05).
Sample
Po osi y (%)
Re
98.3 ± 0.1 c
Ch
98.6 ± 0.1 b
G
99.5 ± 0.1 a
3.1.5. Func ional Cha ac e iza ion
Table 4 shows he esul s ob ained o his s udy ega ding he an ioxidan abili y o
he di e en ae ogels syn hesized. I can be obse ed ha all he ae ogels possess simila
alues associa ed wi h he pe cen age o he inhibi ion alues, showing how hey a e ca-
pable o esis ing oxida ion in a way be e han he whi e sample; he ae ogels con ibu e
o he educ ion in eac i e oxygen species, which can be ound in he wound and which
can p omo e he p oli e a ion o in lamma o y cells. Thus, in his way, i would be possi-
ble o o e come he in lamma o y phase and achie e as e wound healing. Howe e , i
he alues displayed in Table 3 a e compa ed, i can be seen ha he inco po a ion o na-
nopa icles agg a a es he an ioxidan capaci y o he ae ogels, gainsaying he esul s ob-
ained by he nanopa icles which show an enhancemen in he inhibi ion pe cen age
[51,80]. An ioxidan capaci y is a complex p ope y which can be possessed by mul iple
ypes o g oups [81]. I is possible ha a compound ha has a g ea e amoun o an ioxi-
dan g oups may no ha e a good an i adical unc ion. The e ec i eness o he com-
pounds depends on mul iple ac o s, such as hei s uc u al chemical p ope ies; hei
empe a u e; he cha ac e is ics o he subs ance ha is oxidized, i s concen a ion, and i s
Figu e 4. Mic og aphs o di e en ae ogels made: (A) e e ence, (B) chemical nanopa icles included,
and (C) g een nanopa icles inco po a ed.
Table 3. Theo e ical po osi y alues o each ae ogel s udied: e e ence (Re ), chemical nanopa icles
included (Ch), and g een nanopa icles included (G ). Di e en le e s mean signi ican di e ences
(p< 0.05).
Sample Po osi y (%)
Re 98.3 ±0.1 c
Ch 98.6 ±0.1 b
G 99.5 ±0.1 a
3.1.5. Func ional Cha ac e iza ion
Table 4shows he esul s ob ained o his s udy ega ding he an ioxidan abili y o he
di e en ae ogels syn hesized. I can be obse ed ha all he ae ogels possess simila alues
associa ed wi h he pe cen age o he inhibi ion alues, showing how hey a e capable o
esis ing oxida ion in a way be e han he whi e sample; he ae ogels con ibu e o he
educ ion in eac i e oxygen species, which can be ound in he wound and which can
p omo e he p oli e a ion o in lamma o y cells. Thus, in his way, i would be possible o
o e come he in lamma o y phase and achie e as e wound healing. Howe e , i he alues
displayed in Table 3a e compa ed, i can be seen ha he inco po a ion o nanopa icles
agg a a es he an ioxidan capaci y o he ae ogels, gainsaying he esul s ob ained by he
nanopa icles which show an enhancemen in he inhibi ion pe cen age [
51
,
80
]. An ioxidan
capaci y is a complex p ope y which can be possessed by mul iple ypes o g oups [
81
].
I is possible ha a compound ha has a g ea e amoun o an ioxidan g oups may no
ha e a good an i adical unc ion. The e ec i eness o he compounds depends on mul iple
ac o s, such as hei s uc u al chemical p ope ies; hei empe a u e; he cha ac e is ics o
he subs ance ha is oxidized, i s concen a ion, and i s loca ion in he sys em (in e acial
