Ci a ion: Ga cía-Ga cía, A.;
Pé ez-Ál a ez, L.; Ruiz-Rubio, L.;
La ea-Sebal, A.; Ma in, C.;
Vilas-Vilela, J.L. Ex usion-Based 3D
P in ing o Pho oc osslinkable
Chi osan Inks. Gels 2024,10, 126.
h ps://doi.o g/10.3390/
gels10020126
Academic Edi o : Bae Hoon Lee
Recei ed: 20 No embe 2023
Re ised: 30 Janua y 2024
Accep ed: 31 Janua y 2024
Published: 4 Feb ua y 2024
Copy igh : © 2024 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/).
gels
A icle
Ex usion-Based 3D P in ing o Pho oc osslinkable Chi osan Inks
Ane Ga cía-Ga cía1,2 , Ley e Pé ez-Ál a ez 1,2,* , Lei e Ruiz-Rubio 1,2 , Asie La ea-Sebal 3,4,5,
Cesa Ma in 3,4 and JoséLuis Vilas-Vilela 1,2
1
G upo de Química Mac omolecula (LABQUIMAC), Depa amen o de Química Física, Facul ad de Ciencia y
Tecnología, Uni e sidad del País Vasco (UPV/EHU), 48940 Leioa, Spain;
[email p o ec ed] (A.G.-G.); lei [email p o ec ed] (L.R.-R.); [email p o ec ed] (J.L.V.-V.)
2BCMa e ials, Basque Cen e o Ma e ials, Applica ions and Nanos uc u es, UPV/EHU Science Pa k,
48940 Leioa, Spain
3Bio isika Ins i u e (UPV/EHU, CSIC), UPV/EHU Science Pa k, 48940 Leioa, Spain;
asie [email p o ec ed] (A.L.-S.); cesa [email p o ec ed] (C.M.)
4Depa men o Biochemis y and Molecula Biology, Facul ad de Ciencia y Tecnología, Uni e sidad del País
Vasco UPV/EHU, 48940 Leioa, Spain
5Fundación Bio isika Bizkaia, Ba io Sa iena s/n., 48940 Leioa, Spain
*Co espondence: ley e.pe [email p o ec ed]; Tel.: +34-946012709
Abs ac : Pho ocu ing o chi osan has shown g ea p omise in he ex usion-based 3D p in ing o
sca olds o ad anced biomedical and issue enginee ing applica ions. Howe e , he poo mechanical
s abili y o me hac yla ed chi osan pho ocu ing ink es ic s i s applicabili y. The inclusion o co-
ne wo ks by means o simul aneous polycomplex o ma ion is an e ec i e me hod by which o
sol e his d awback, bu he o med hyd ogel inks a e no p in able. This wo k aims o de elop
new pho ocu able chi osan inks based on he simul aneous pho oc osslinking o me hac yla ed
chi osan (CHIMe) wi h N,N
′
-me hylenebisac ylamide, polye hylene glycol diac yla e, and ac ylic
acid o be applied in ex usion 3D p in ing. In e es ingly, he polycomplex co-ne wo k co esponding
o he ac ylic-acid-based ink could be success ully p in ed by he he e-p esen ed simul aneous
pho ocu ing s a egy. Fu he , he con e sion o pho oc osslinking was s udied ia pho o-DSC
analyses ha e ealed a clea dependence on he chemical s uc u e o he employed c osslinking
agen s ( om 40 o ~100%). In addi ion, he mechanical and heological p ope ies o he pho ocu ed
hyd ogels we e compa a i ely s udied, as well as he p in ing quali y o he ex uded sca olds. The
newly de eloped chi osan pho ocu able inks demons a ed ex usion p in abili y (squa eness ~0.90;
uni o mi y ac o ~0.95) and unable mechanical p ope ies (Young modulus 14–1068 Pa) by means o
di e en c osslinking app oaches acco ding o he chemical a chi ec u e o he eac i e molecules
employed. This wo k shows he g ea po en ial o pho oc osslinkable chi osan inks.
Keywo ds: me hac yla ed chi osan; pho oc osslinking; ex usion p in ing
1. In oduc ion
Tissue enginee ing and egene a i e medicine, which a e e ol ing ields based on
he de elopmen o new bioma e ials, ha e signi ican ly p og essed due o he ecen
ad ance o 3D p in ing echniques. This is based on he ac ha issue de elopmen and
egene a ion s a egies equi e biocompa ible 3D sca olds ha ac as an ex acellula ma ix
(ECM), p o iding cells wi h pe sonalized 3D a chi ec u es and se ing as a con olled
s imuli sou ce o he egula ion o cell g ow h, p oli e a ion, and di e en ia ion [
1
]. Th ee-
dimensional p in ing consis s o he h ee-dimensional deposi ion and sequen ial laye ing
o ma e ials using a compu e -c ea ed design. This addi i e p ocess has opened up new
possibili ies o designing pe sonalized sca olds wi h high p ecision, ep oducibili y, and
adap abili y in compa ison wi h mo e adi ional me hods like eplica molding.
Gels 2024,10, 126. h ps://doi.o g/10.3390/gels10020126 h ps://www.mdpi.com/jou nal/gels
Gels 2024,10, 126 2 o 16
In he con ex o an escala ing in e es in 3D-p in able bioma e ials, no el ink o mu-
la ions wi h selec i e biological ea u es, high p in ing ideli y, s abili y, and app op ia e
mechanical p ope ies a e highly demanded [2].
Among he 3D p in ing echniques cu en ly used o issue enginee ing applica ions,
ex usion-based p in ing is ecognized as a simple addi i e manu ac u ing echnique ha
in ol es he ex usion o cy ocompa ible ma e ials able o mimic he ECM en i onmen
h ough mic ome ic nozzles o ab ica e h ee-dimensional s uc u es [
3
]. Despi e i s
low esolu ion in compa ison wi h lase -media ed o s e eoli hog aphic app oaches, ex-
usion p in ing, due o i s simplici y, is he mos -used echnique o he 3D p in ing o
polyme hyd ogels [3,4].
Hyd ogels a e hyd ophilic polyme ne wo ks, ecen ly exploi ed as inks wi h which
o ab ica e sca olds due o hei esemblance o an ex acellula ma ix (ECM) and hei
abili y o acili a e and egula e cell mig a ion and adhesion [
2
]. Hyd ogel elas ome ic
ne wo ks a e o med ia he physical o chemical c osslinking o polyme chains wi h
hemsel es and/o wi h mul i unc ional eac i e c osslinking agen s [5].
In ex usion-based hyd ogels p in ing, as he liquid ink is ex uded ou o a ca idge,
he deposi ed iscous gel is p in ed as long as i s heological p ope ies a e app op ia e
o main aining mechanical s abili y and shape ideli y [
6
]. To enable his, oge he wi h
deposi ion, a phase ansi ion akes place as a consequence o he physical and/o chemical
c osslinking o he polyme ic chains [
7
]. Among he di e en s imuli ha allow o his
sol–gel ansi ion o he hyd ogel ink [
8
], i is wo h highligh ing he widely exploi ed
iono opic gela ion o algina e biopolyme [
9
,
10
]. Howe e , ligh i adia ion as he s imuli,
and pho ocu ing as a mechanism, ha e eme ged as p omising app oaches o p omo ing
as and s able chemical c osslinks, ensu ing he highe esolu ion equi ed o he p in ing
o complex s uc u es.
The mos ypical pho ocu ing eac ions in he biomedical ield, oge he wi h hiol–ene
eac ions, a e ee adical chain eac ions [
11
], which a e usually based on inyl syn-
he ic monome s and oligome s ha polyme ize h ough a chain g ow h mechanism, o
inyl polyme s ha a e chemically c osslinked [
12
]. Solu ions o (me h)ac yla e-based
monome s/oligome s/polyme s a e widely employed as 3D pho ocu ing adical inks
because hey a e highly compa ible wi h comme cially a ailable 3D p in e s [
13
]. Al hough
me hac yla es a e less eac i e han ac yla es, hey a e highly p e e ed in biomedical
applica ions since hey display much lowe oxici y [
14
], which has p o oked he ecen
ou b eak o in es iga ions in o me hac yla ed de i a i es as pho ocu able bioma e ials [
12
].
Al hough a signi ican ad ance has been made in he pho ocu ing o hyd ogels, as
is he case wi h he success ul 3D p in ing o comme cial me hac yla ed algina e and
me hac yla ed gela ine-based o mula ions, cu en ly, he e is s ill a eal need o a la ge
a ie y o p in able hyd ogel inks o sp ead ou he p og ess in his ield [15].
In he sea ch o new p in able inks o issue enginee ing pu poses, na u ally de-
i ed hyd ogels, ha a e o iginal ma e ials om an ex acellula ma ix, biocompa ible,
biodeg adable unde physiological condi ions, and no oxic, ha e shown o be excellen
candida es [
16
]. Polysaccha ides a e speci ically in e es ing as a na u al, sus ainable, and
enewable sou ce o bioma e ials, and consequen ly, cu en esea ch on na u al–hyd ogel
inks has ocused on hese biopolyme s [
10
]. Limi a ions associa ed wi h some polysaccha-
ides, such as he high iscosi y o aga ose o he subop imal cell a achmen and p o ein
abso p ion cha ac e is ics o algina e, ha e p omp ed he explo a ion and u iliza ion o
al e na i e biopolyme s.
Chi osan is a na u ally de i ed polysaccha ide o en used in issue enginee ing since,
apa om being biocompa ible, non oxic, simila o an ex acellula ma ix, i deg ades
o oligome s ia lysozyme, which is p esen in he human body. In addi ion, chi osan
p esen s unique biological p ope ies, such as i s an imic obial ac i i y, ha ha e d i en
he g ow h o chi osan de i a i es in all hei possible o ms [
17
]. In ela ion o he appli-
cabili y o his polysaccha ide as a 3D p in ing bioink, chi osan solu ions no only exhibi
s abili y unde physiological condi ions and sui able iscosi y alues o biop in ing ap-
Gels 2024,10, 126 3 o 16
plica ions; hey a e also conduci e o p ope cell p oli e a ion and di e en ia ion. All his
means ha chi osan-based inks a e posi ioned a he o e on o candida e inks o 3D
biop in ing applica ions due o hei wide iscosi y ange, di e se c osslinking mechanisms,
and adjus able mechanical p ope ies, oge he wi h hei ema kable cell iabili y and
an ibac e ial ac i i y [18].
In he con ex o chi osan c osslinking, i unde goes gela ion h ough bo h physical and
chemical c oss-linking mechanisms. Howe e , one no able d awback o issue enginee ing
applica ions is he inhe en ly slow gela ion a e associa ed wi h hese mechanisms. This
limi a ion con as s wi h he as e p ocess o pho ocu ing. In i s o iginal o m, chi osan is
no inhe en ly pho ocu able. Ne e heless, ecen ad ancemen s in he pas decade ha e
explo ed he use o UV ligh i adia ion o chi osan gela ion, which is achie ed h ough
p io modi ica ion ia me hac yla ion eac ions. This modi ied app oach p esen s a mo e
e icien and apid c osslinking me hod o chi osan, add essing he ime- ela ed challenges
associa ed wi h adi ional gela ion mechanisms and enhancing i s sui abili y o applica-
ions in issue enginee ing. [
19
]. Me hac yla ed chi osan has shown he abili y o quickly
o m mechanically s able ne wo ks ia an in si u pho ocu ing p ocess. Indeed, he e a e se -
e al examples o s udies ha employ me hac yla ed chi osan o biomedical
pu poses [20]
.
Ne e heless, despi e he al eady- epo ed in es iga ions on pho oc osslinkable chi osan
sca olds [
21
], 3D p in ing o s able chi osan-based s uc u es is s ill a challenging issue ha
is unde ea ly explo a ion [22].
The main eason behind his is he poo mechanical s eng h o he singula biopoly-
me hyd ogel. To o e come his d awback, he addi ion o mul i unc ional ac yla e
monome s [
14
] o polyme s [
23
] ac ing as co alen c osslinking agen s and o ming a
double-componen ink is commonly applied in hese cases.
The inco po a ion o mul i unc ional monome s ac ing as c osslinking agen s is an
in e es ing al e na i e because hey p omo e apid cu ing and good spa ial esolu ion. How-
e e , b i leness and high c osslink densi y de i ed om hese ypes o c osslinking agen s
a e se ious issues ha es ic hei applicabili y, especially in he biomedical ield [14].
Among syn he ic hyd ogels, pho ocu ed polye hylene glycol diac yla e (PEGDA)
hyd ogels ha e been widely in es iga ed in issue enginee ing due o hei hyd ophilici y,
high mechanical s abili y, biocompa ibili y, and biodeg adabili y. Wi h his in mind, plen y
o examples ha e a isen in he bibliog aphy ha ake ad an age o he bene i s o his
syn he ic polyme o egula e he mechanical p ope ies, deg ada ion a e, and/o p in -
abili y o me hac yla ed na u al polyme s by p epa ing chemically o med co-ne wo ks by
eac ion wi h PEGDA [24,25].
Physical co-ne wo ks o pho ocu ed chi osan, esul ing om addi ional physical o ces
such as hyd ogen bonding o elec os a ic in e ac ions, ha e also been explo ed o enhance
he mechanical p ope ies o pho oc osslinkable me hac yla ed chi osan [
26
]. In e es ingly,
chi osan is able o o m polycomplexes wi h na u al o syn he ic polyacids in solu ion
ia spon aneous associa ion, leading o he o ma ion o s ong polycomplex ne wo ks
wi hou equi ing he use o chemical c osslinking agen s [
27
]. The combina ion o chi osan–
polyacid polycomplexes wi hin a pho oc osslinked chi osan hyd ogel esul s in doubly
in e pene a ed ne wo ks ha ha e demons a ed ailo ed s i ness and deg adabili y.
Howe e , despi e some o hese chi osan–polyacid polycomplexes ul illing he main
equi emen s o be ex uded, hei high cohesi e o ces limi hei di ec p in ing abili y,
which has been shown o be es ic ed o a mul ima e ial and low-quali y laye ia he laye
p in ing p ocess [28].
In his con ex , his wo k explo es he po en ial pho o-induced p in ing o a new o mu-
la ion o me hac yla ed chi osan ink, including acid and pho opolyme izable monome ic
uni s ha can polyme ize du ing pho ocu ing in combina ion wi h chi osan pho oc osslink-
ing leading o physical co-ne wo ks. This wo k’s hypo hesis is based on he easibili y
o he accu a e p in ing o he in e pene a ed ne wo k o me hac yla e chi osan and he
polyacid, which is suppo ed by he ac ha he polycomplex o ma ion be ween chi-
osan and he polyacid akes place a e gel deposi ion and while he pho oc osslinking
Gels 2024,10, 126 4 o 16
p ocess o he bulk me hac yla ed ne wo k akes place. Fo his, ac ylic acid was cho-
sen as an acidic monome ic p ecu so o polycomplex o ma ion wi h me hac yla ed
chi osan. The pho op in ing abili y and p in ing quali y o ac ylic acid-based ink we e
compa ed wi h hose o he plain me hac yla ed chi osan ink and wi h wo di e en co-
alen ly o med co-ne wo ks inks. The p oposed co alen co-ne wo ks we e based on
he combina ion o me hac yla ed chi osan wi h PEGDA, a mul i unc ional c osslinking
polyme , and N,N
′
-me hylenebisac ylamide (NMBA), a mul i unc ional and biocompa ible
c osslinking monome , which a e adi ionally employed as c osslinking agen in bulk
hyd ogels (Figu e 1). Mo eo e , heological and mechanical p ope ies o pho ocu ed
hyd ogels we e also compa a i ely analyzed.
Gels 2024, 10, x FOR PEER REVIEW 4 o 17
chi osan and he polyacid akes place a e gel deposi ion and while he pho oc osslinking
p ocess o he bulk me hac yla ed ne wo k akes place. Fo his, ac ylic acid was chosen
as an acidic monome ic p ecu so o polycomplex o ma ion wi h me hac yla ed chi-
osan. The pho op in ing abili y and p in ing quali y o ac ylic acid-based ink we e com-
pa ed wi h hose o he plain me hac yla ed chi osan ink and wi h wo diffe en co alen ly
o med co-ne wo ks inks. The p oposed co alen co-ne wo ks we e based on he combi-
na ion o me hac yla ed chi osan wi h PEGDA, a mul i unc ional c osslinking polyme ,
and N,N’-me hylenebisac ylamide (NMBA), a mul i unc ional and biocompa ible c oss-
linking monome , which a e adi ionally employed as c osslinking agen in bulk hyd o-
gels (Figu e 1). Mo eo e , heological and mechanical p ope ies o pho ocu ed hyd ogels
we e also compa a i ely analyzed.
Figu e 1. A ep esen a i e scheme o he syn he ic app oach o he p epa a ion o me hac yla ed
chi osan hyd ogels wi h NMBA, PEG, and AA as c osslinking agen s.
2. Resul s and Discussion
2.1. Hyd ogels Pho ocu ing
P is ine chi osan was modi ied by eac ion wi h me hac ylic anhyd ide in o de o
become pho oc osslinkable; hus, i s solu ion can ac as a pho ocu able ink a e he pho-
oini ia o addi ion. This modi ica ion eac ion akes place ia he nucleophilic a ack o
he amine g oup in chi osan on he ca bonyl g oup in me hac ylic anhyd ide, gi ing me h-
ac yla ed chi osan as a p oduc . The success ul syn hesis was con i med ia 1H-NMR spec-
oscopy (Figu e 2).
Figu e 1. A ep esen a i e scheme o he syn he ic app oach o he p epa a ion o me hac yla ed
chi osan hyd ogels wi h NMBA, PEG, and AA as c osslinking agen s.
2. Resul s and Discussion
2.1. Hyd ogels Pho ocu ing
P is ine chi osan was modi ied by eac ion wi h me hac ylic anhyd ide in o de o
become pho oc osslinkable; hus, i s solu ion can ac as a pho ocu able ink a e he pho-
oini ia o addi ion. This modi ica ion eac ion akes place ia he nucleophilic a ack
o he amine g oup in chi osan on he ca bonyl g oup in me hac ylic anhyd ide, gi ing
me hac yla ed chi osan as a p oduc . The success ul syn hesis was con i med ia
1
H-NMR
spec oscopy (Figu e 2).
Gels 2024, 10, x FOR PEER REVIEW 5 o 17
Figu e 2. 1H-NMR spec a o (a) p is ine chi osan and (b) me hac yla ed chi osan.
In he 1H-NMR spec a o chi osan (Figu e 2a), in addi ion o he signals o he me hyl
p o ons o he ace yl g oup and he deace yla ed p o on a 2 and 2.8 ppm, espec i ely,
he signals co esponding o he p o ons in he glucosamine ing can be seen be ween 2.8
and 3.9 ppm. In he 1H-NMR spec a o me hac yla ed chi osan (Figu e 2b), hese same
signals a e obse ed, bu also hose ha appea ed a 5.6–6.2 ppm, asc ibed o he p o ons
o he alkenyl g oup in he me hac yla e moie y. Mo eo e , a 1.85 ppm, a signal co e-
sponding o he p o ons in he me hyl g oup o he me hac yla e moie y can also be ob-
se ed [29]. In addi ion o quali a i ely co obo a ing he success o he modi ica ion e-
ac ion o chi osan, 1H-NMR spec oscopy allowed o he de e mina ion o he me hac y-
la ion deg ee. This was calcula ed ia he in eg a ion o he p o ons in he alkenyl g oup
(Ha, Hb) wi h espec o he in eg a ion o he p o ons in he glucosamine ing, esul ing
in an a e age me hac yla ion deg ee o 54 ± 9% ha enables he u he pho oc osslinking
o chi osan.
LAP pho o-ini ia o was selec ed due o i s high wa e solubili y and isible ligh -
sensi i e pho oini ia ion (405 nm), which leads o high cell iabili y [30]. Hyd ogel inks
we e p epa ed by mixing me hac yla ed chi osan acidic solu ion wi h pho oini ia o solu-
ion and, in he case o AA, NMBA, and PEGDA, co-ne wo ks, by adding he co espond-
ing amoun o c osslinking agen as desc ibed in he Expe imen al Sec ion.
The con e sion (α) o he pho oc osslinking o me hac yla ed chi osan in he p es-
ence o he diffe en inyl compounds was analyzed ia pho o-DSC in o de o ob ain
in o ma ion abou he pho op in ing abili y o he inks (Figu e 3).
Figu e 2. 1H-NMR spec a o (a) p is ine chi osan and (b) me hac yla ed chi osan.
Gels 2024,10, 126 5 o 16
In he
1
H-NMR spec a o chi osan (Figu e 2a), in addi ion o he signals o he me hyl
p o ons o he ace yl g oup and he deace yla ed p o on a 2 and 2.8 ppm, espec i ely, he
signals co esponding o he p o ons in he glucosamine ing can be seen be ween 2.8 and
3.9 ppm. In he
1
H-NMR spec a o me hac yla ed chi osan (Figu e 2b), hese same signals
a e obse ed, bu also hose ha appea ed a 5.6–6.2 ppm, asc ibed o he p o ons o he
alkenyl g oup in he me hac yla e moie y. Mo eo e , a 1.85 ppm, a signal co esponding
o he p o ons in he me hyl g oup o he me hac yla e moie y can also be obse ed [
29
]. In
addi ion o quali a i ely co obo a ing he success o he modi ica ion eac ion o chi osan,
1
H-NMR spec oscopy allowed o he de e mina ion o he me hac yla ion deg ee. This
was calcula ed ia he in eg a ion o he p o ons in he alkenyl g oup (Ha, Hb) wi h
espec o he in eg a ion o he p o ons in he glucosamine ing, esul ing in an a e age
me hac yla ion deg ee o 54 ±9% ha enables he u he pho oc osslinking o chi osan.
LAP pho o-ini ia o was selec ed due o i s high wa e solubili y and isible ligh -
sensi i e pho oini ia ion (405 nm), which leads o high cell iabili y [
30
]. Hyd ogel inks
we e p epa ed by mixing me hac yla ed chi osan acidic solu ion wi h pho oini ia o solu-
ion and, in he case o AA, NMBA, and PEGDA, co-ne wo ks, by adding he co esponding
amoun o c osslinking agen as desc ibed in he Expe imen al Sec ion.
The con e sion (
α
) o he pho oc osslinking o me hac yla ed chi osan in he p es-
ence o he di e en inyl compounds was analyzed ia pho o-DSC in o de o ob ain
in o ma ion abou he pho op in ing abili y o he inks (Figu e 3).
Gels 2024, 10, x FOR PEER REVIEW 6 o 17
Figu e 3. Pho o-c osslinking con e sion o (black) p is ine me hac yla ed chi osan, and me hac y-
la ed chi osan wi h ( ed) polye hyleneglycol diac yla e, (blue) N,N’-me hylenebisac ylamide, and
(g een) ac ylic acid.
Rega ding he con e sion ime, i can be obse ed ha all he samples a e cu ed in
unde ou minu es, i.e., imes ha a e sho enough o po en ially limi he sp eading and
which a o pho ocu ing-media ed ex usion p in ing. In compa ison wi h me hac yla ed
chi osan, i can be obse ed ha he inco po a ion o addi ional inyl compounds im-
p o es he po en ial p in abili y o he inks, leading o highe con e sion alues and lowe
cu ing imes. The pho o-c osslinking s udy e eals an in e es ing ole o he chemical a -
chi ec u e o he employed inyl molecules. This could be ela ed o he mobili y o he
eac ing molecules, which, in u n, is closely linked o he inal eac i i y o he ink. In he
absence o ex e nal agen s, inyl moie ies a e p esen along he chains o high-molecula -
weigh chi osan chains (highly iscous chi osan), which p esen educed mobili y; conse-
quen ly, poo con e sion alues a e egis e ed. Howe e , when smalle bi unc ional mol-
ecules ac ing as c osslinking agen s, like NMBA and PEGDA, a e added, eac i i y aises,
leading o a g ea inc ease in con e sion (~2 imes) and a educ ion in cu ing ime (~2
imes). I is wo h highligh ing he good pe o mance o PEGDA, which can be explained
by he well-known lexibili y o he linea polye hylene glycol chains. When unc ionali y
is aken in o conside a ion, mul i unc ional monome s show ha e mina ion eac ions
a e mobili y es ic ed, esul ing in he au oaccele a ion o he pho ocu ing [14]. The pho-
ocu ing cu e o he ink ha p esen s mono unc ional ac ylic acid is also in line wi h his
explana ion. Acco dingly, i s mono unc ional na u e leads o a lowe con e sion alue
han when di unc ional c osslinke s a e employed, o which e mina ion eac ions a e
mobili y es ic ed [14].
2.2. Mo phology
Figu e 4 shows he su ace mo phology o me hac yla ed chi osan hyd ogels p e-
pa ed wi h diffe en c osslinkable agen s pho og aphed ia scanning elec on mic oscopy
(SEM). In all samples, po ous and in e connec ed 3D ne wo ks wi h ela i ely uni o m
po e size dis ibu ions could be obse ed, which is o g ea in e es o biological applica-
ions. The ob ained po e size alues we e also ep esen a i e o he molecula s uc u e o
he employed inyl molecules, showing a signi ican dec ease wi h he addi ion o ac ylic
acid and NMBA, while la ge po e sizes we e measu ed in he hyd ogel c osslinked wi h
PEGDA. Acco ding o he esul s, i mus be highligh ed ha he po e size o PEGDA co-
ne wo ks seems o be go e ned by he lexible and hyd ophilic s uc u e o PEGDA in-
s ead o he highe c osslinking densi y de i ed om a highe measu ed con e sion. Thus,
he swellable s uc u e esul ing om PEGDA-media ed c osslinking a o s highe wa e
up ake wi hin hyd ogels, leading, a e sample eeze d ying, o highe po e sizes han in
he case o p is ine CHIMe ne wo k. Acco ding o his, igid and sho -leng h NMBA
Figu e 3. Pho o-c osslinking con e sion o (black) p is ine me hac yla ed chi osan, and me hac yla ed
chi osan wi h ( ed) polye hyleneglycol diac yla e, (blue) N,N
′
-me hylenebisac ylamide, and (g een)
ac ylic acid.
Rega ding he con e sion ime, i can be obse ed ha all he samples a e cu ed in
unde ou minu es, i.e., imes ha a e sho enough o po en ially limi he sp eading and
which a o pho ocu ing-media ed ex usion p in ing. In compa ison wi h me hac yla ed
chi osan, i can be obse ed ha he inco po a ion o addi ional inyl compounds imp o es
he po en ial p in abili y o he inks, leading o highe con e sion alues and lowe cu ing
imes. The pho o-c osslinking s udy e eals an in e es ing ole o he chemical a chi ec u e
o he employed inyl molecules. This could be ela ed o he mobili y o he eac ing
molecules, which, in u n, is closely linked o he inal eac i i y o he ink. In he absence
o ex e nal agen s, inyl moie ies a e p esen along he chains o high-molecula -weigh
chi osan chains (highly iscous chi osan), which p esen educed mobili y; consequen ly,
poo con e sion alues a e egis e ed. Howe e , when smalle bi unc ional molecules
ac ing as c osslinking agen s, like NMBA and PEGDA, a e added, eac i i y aises, leading
o a g ea inc ease in con e sion (~2 imes) and a educ ion in cu ing ime (~2 imes).
I is wo h highligh ing he good pe o mance o PEGDA, which can be explained by
he well-known lexibili y o he linea polye hylene glycol chains. When unc ionali y
is aken in o conside a ion, mul i unc ional monome s show ha e mina ion eac ions
Gels 2024,10, 126 6 o 16
a e mobili y es ic ed, esul ing in he au oaccele a ion o he pho ocu ing [
14
]. The
pho ocu ing cu e o he ink ha p esen s mono unc ional ac ylic acid is also in line wi h
his explana ion. Acco dingly, i s mono unc ional na u e leads o a lowe con e sion alue
han when di unc ional c osslinke s a e employed, o which e mina ion eac ions a e
mobili y es ic ed [14].
2.2. Mo phology
Figu e 4shows he su ace mo phology o me hac yla ed chi osan hyd ogels p epa ed
wi h di e en c osslinkable agen s pho og aphed ia scanning elec on mic oscopy (SEM).
In all samples, po ous and in e connec ed 3D ne wo ks wi h ela i ely uni o m po e
size dis ibu ions could be obse ed, which is o g ea in e es o biological applica ions.
The ob ained po e size alues we e also ep esen a i e o he molecula s uc u e o he
employed inyl molecules, showing a signi ican dec ease wi h he addi ion o ac ylic
acid and NMBA, while la ge po e sizes we e measu ed in he hyd ogel c osslinked wi h
PEGDA. Acco ding o he esul s, i mus be highligh ed ha he po e size o PEGDA co-
ne wo ks seems o be go e ned by he lexible and hyd ophilic s uc u e o PEGDA ins ead
o he highe c osslinking densi y de i ed om a highe measu ed con e sion. Thus,
he swellable s uc u e esul ing om PEGDA-media ed c osslinking a o s highe wa e
up ake wi hin hyd ogels, leading, a e sample eeze d ying, o highe po e sizes han
in he case o p is ine CHIMe ne wo k. Acco ding o his, igid and sho -leng h NMBA
c osslinking agen p omo es smalle po e sizes in he CHIMe-NMBA hyd ogels, which is
in acco dance wi h a e age measu ed alue in hese samples in compa ison wi h he po e
size alue o he simple CHIMe hyd ogel. In e es ingly, as can be obse ed in Figu e 4c, he
inco po a ion o AA in he ink leads o an impo an dec ease in he size o po es. This e ec
is explained by he o ma ion o he doubly in e pene a ed ne wo k consequence o he
complexa ion o chi osan wi h he polyacid. In his sense, he polycomplexa ion s a egy
wi h AA u ns ou o be a mo e e ec i e in es ic ing po e size in chi osan pho oc osslinked
hyd ogels han c osslinking wi h NMBA.
Gels 2024, 10, x FOR PEER REVIEW 7 o 17
c osslinking agen p omo es smalle po e sizes in he CHIMe-NMBA hyd ogels, which is
in acco dance wi h a e age measu ed alue in hese samples in compa ison wi h he po e
size alue o he simple CHIMe hyd ogel. In e es ingly, as can be obse ed in Figu e 4c,
he inco po a ion o AA in he ink leads o an impo an dec ease in he size o po es. This
effec is explained by he o ma ion o he doubly in e pene a ed ne wo k consequence
o he complexa ion o chi osan wi h he polyacid. In his sense, he polycomplexa ion
s a egy wi h AA u ns ou o be a mo e effec i e in es ic ing po e size in chi osan pho-
oc osslinked hyd ogels han c osslinking wi h NMBA.
Figu e 4. Rep esen a i e SEM mic og aphs o CHIMe (a), CHIMe-PEGDA (b), CHIMe-AA (c), and
CHIMe-NMBA (d) hyd ogels.
2.3. Rheological and Mechaniccal P ope ies
The heological p ope ies o he CHIMe inks p epa ed wi h and wi hou he ana-
lyzed inyl compounds we e s udied by means o equency sweep measu emen s. The
esul s ob ained in Figu e 5 show ha all he me hac yla ed chi osan samples show highe
alues in s o age modulus han in loss modulus (G′ > G″) h ough he en i e equency
ange. This beha io means ha he elas ic p ope ies o he analyzed samples ha e a
g ea e effec han he iscose ones, which is a ypical beha io o s able hyd ogels.
Figu e 4. Rep esen a i e SEM mic og aphs o CHIMe (a), CHIMe-PEGDA (b), CHIMe-AA (c), and
CHIMe-NMBA (d) hyd ogels.
Gels 2024,10, 126 7 o 16
2.3. Rheological and Mechaniccal P ope ies
The heological p ope ies o he CHIMe inks p epa ed wi h and wi hou he analyzed
inyl compounds we e s udied by means o equency sweep measu emen s. The esul s
ob ained in Figu e 5show ha all he me hac yla ed chi osan samples show highe alues
in s o age modulus han in loss modulus (G
′
> G
′′
) h ough he en i e equency ange.
This beha io means ha he elas ic p ope ies o he analyzed samples ha e a g ea e
e ec han he iscose ones, which is a ypical beha io o s able hyd ogels.
Gels 2024, 10, x FOR PEER REVIEW 8 o 17
Figu e 5. S o age (G′, illed ci cles) and loss module (G″, open ci cles) o me hac yla ed chi osan
wi h NMBA, PEG, and AA as c osslinking agen s (1% s ain).
I is no ewo hy ha he inco po a ion o an ex e nal inyl agen leads o an inc ease
in he s o age modulus o he hyd ogels (CHIMe-PEGDA, CHIMe-NMBA, CHIMe-AA)
wi h espec o he p is ine me hac yla ed chi osan. No ably, a co ela ion be ween he
s o age moduli o he hyd ogels and he pho ocu ing con e sion yield de e mined by
pho o-DSC measu emen s can be also obse ed. Indeed, highe s o age moduli co e-
spond o hyd ogel samples whichp esen highe pho ocu ing con e sion ha can be as-
c ibed o a g ea e c osslinking densi y and, consequen ly, o a mo e solid-like beha io .
S ess–s ain comp ession cu es we e also ob ained o each hyd ogel ype, as can
be seen in Figu e 6. In acco dance wi h heological esul s, a g ea imp o emen in he
mechanical p ope ies can be app ecia ed when ex e nal c osslinking agen s we e added
in compa ison o CHIMe samples wi hou addi ional c osslinke s. I is wo h highligh ing
he lowe ac u e s ain shown by he hyd ogels c osslinked wi h NMBA. This b i leness
seems o be ela ed o he sho leng h and igidi y cha ac e is ic o his c osslinking agen
[14]. Indeed, NMBA is ypically employed as a success ul c osslinke in polycac ylamides
hyd ogels. These hyd ogels a e cha ac e ized by hei own high elas ici y, unlike in he
case o he he e-employed me hac yla ed chi osan chains. The inhe en elas ici y o
polyac ylamide hyd ogels coun e ac s he igid na u e o he NMBA c osslinking agen ,
as is obse ed in me hac yla ed chi osan ne wo ks.
Figu e 5. S o age (G
′
, illed ci cles) and loss module (G
′′
, open ci cles) o me hac yla ed chi osan wi h
NMBA, PEG, and AA as c osslinking agen s (1% s ain).
I is no ewo hy ha he inco po a ion o an ex e nal inyl agen leads o an inc ease in
he s o age modulus o he hyd ogels (CHIMe-PEGDA, CHIMe-NMBA, CHIMe-AA) wi h
espec o he p is ine me hac yla ed chi osan. No ably, a co ela ion be ween he s o age
moduli o he hyd ogels and he pho ocu ing con e sion yield de e mined by pho o-DSC
measu emen s can be also obse ed. Indeed, highe s o age moduli co espond o hyd ogel
samples whichp esen highe pho ocu ing con e sion ha can be asc ibed o a g ea e
c osslinking densi y and, consequen ly, o a mo e solid-like beha io .
S ess–s ain comp ession cu es we e also ob ained o each hyd ogel ype, as can
be seen in Figu e 6. In acco dance wi h heological esul s, a g ea imp o emen in he
mechanical p ope ies can be app ecia ed when ex e nal c osslinking agen s we e added in
compa ison o CHIMe samples wi hou addi ional c osslinke s. I is wo h highligh ing
he lowe ac u e s ain shown by he hyd ogels c osslinked wi h NMBA. This b i le-
ness seems o be ela ed o he sho leng h and igidi y cha ac e is ic o his c osslinking
agen [
14
]. Indeed, NMBA is ypically employed as a success ul c osslinke in polycac y-
lamides hyd ogels. These hyd ogels a e cha ac e ized by hei own high elas ici y, unlike
in he case o he he e-employed me hac yla ed chi osan chains. The inhe en elas ici y o
polyac ylamide hyd ogels coun e ac s he igid na u e o he NMBA c osslinking agen , as
is obse ed in me hac yla ed chi osan ne wo ks.
Gels 2024,10, 126 8 o 16
Gels 2024, 10, x FOR PEER REVIEW 9 o 17
Figu e 6. Mechanical s abili y o me hac yla ed chi osan wi h and wi hou c osslinke s unde com-
p ession s ess/s ain es s.
2.4. Ex usion 3D P in ing
Nozzle diame e , p in ing speed and p in ing p essu e we e op imized o
me hac yla ed chi osan ink and he esul s a e shown in Figu e 7. I can be seen ha he
expansion a io dec eases wi h dec easing p essu e and inc easing speed, while he
uni o mi y ac o is close o 1 o low p essu e and dec easing speed. Acco dingly, 10 kPa
and 10 mm/s we e selec ed as he mos accu a e p essu e and speed p in ing condi ions
o all he analyzed me hac yla ed chi osan-based inks.
Figu e 6. Mechanical s abili y o me hac yla ed chi osan wi h and wi hou c osslinke s unde
comp ession s ess/s ain es s.
2.4. Ex usion 3D P in ing
Nozzle diame e , p in ing speed and p in ing p essu e we e op imized o me hac y-
la ed chi osan ink and he esul s a e shown in Figu e 7. I can be seen ha he expansion
a io dec eases wi h dec easing p essu e and inc easing speed, while he uni o mi y ac o
is close o 1 o low p essu e and dec easing speed. Acco dingly, 10 kPa and 10 mm/s we e
selec ed as he mos accu a e p essu e and speed p in ing condi ions o all he analyzed
me hac yla ed chi osan-based inks.
Gels 2024, 10, x FOR PEER REVIEW 9 o 17
Figu e 6. Mechanical s abili y o me hac yla ed chi osan wi h and wi hou c osslinke s unde com-
p ession s ess/s ain es s.
2.4. Ex usion 3D P in ing
Nozzle diame e , p in ing speed and p in ing p essu e we e op imized o
me hac yla ed chi osan ink and he esul s a e shown in Figu e 7. I can be seen ha he
expansion a io dec eases wi h dec easing p essu e and inc easing speed, while he
uni o mi y ac o is close o 1 o low p essu e and dec easing speed. Acco dingly, 10 kPa
and 10 mm/s we e selec ed as he mos accu a e p essu e and speed p in ing condi ions
o all he analyzed me hac yla ed chi osan-based inks.
Figu e 7. E ec o ex usion p essu e (10, 15, and 20 kPa) and p in ing speed (5, 10, and 13.3 mm/s)
on (a) uni o mi y ac o , and (b) expansion a io o me hac yla ed chi osan ink p in ing using a nozzle
diame e o 0.20 mm o me hac yla ed chi osan.
Gels 2024,10, 126 9 o 16
Wi h he pu pose o s udying he e ec o he inco po a ion o selec ed inyl com-
pounds on he p in abili y o me hac yla ed chi osan ink, squa e-shaped sca olds we e
p in ed (8
×
8 po e pe side wi h an a ea o 0.25 cm
2
o each po e) ollowing he abo e op-
imized condi ions and a ying he ligh in ensi y (20 and 190 W/m
2
). Op ical mic oscope
images o he esul ing sca olds a e shown in Figu e 8. As can be obse ed (Figu e 8), he
ink co esponding o he doubly c osslinked polycomplex (CHIMe-AA) could no only be
accu a ely p in ed ollowing he same p in ing condi ions as CHIMe p is ine ink; i also
displayed a highe p in ing quali y.
Gels 2024, 10, x FOR PEER REVIEW 10 o 17
Figu e 7. Effec o ex usion p essu e (10, 15, and 20 kPa) and p in ing speed (5, 10, and 13.3 mm/s)
on (a) uni o mi y ac o , and (b) expansion a io o me hac yla ed chi osan ink p in ing using a noz-
zle diame e o 0.20 mm o me hac yla ed chi osan.
Wi h he pu pose o s udying he effec o he inco po a ion o selec ed inyl
compounds on he p in abili y o me hac yla ed chi osan ink, squa e-shaped scaffolds
we e p in ed (8 × 8 po e pe side wi h an a ea o 0.25 cm2 o each po e) ollowing he
abo e op imized condi ions and a ying he ligh in ensi y (20 and 190 W/m2). Op ical
mic oscope images o he esul ing scaffolds a e shown in Figu e 8. As can be obse ed
(Figu e 8), he ink co esponding o he doubly c osslinked polycomplex (CHIMe-AA)
could no only be accu a ely p in ed ollowing he same p in ing condi ions as CHIMe
p is ine ink; i also displayed a highe p in ing quali y.
Figu e 8. Op ical mic oscope images o 8 × 8 squa e scaffolds o CHIMe, CHIMe-NMBA, CHIMe-
PEG, and CHIMe-AA p in ed unde 405 nm and 190 W/m2 ligh .
Acco ding o Figu e 9, he addi ion o ex e nal c osslinking in CHIMe ink leads o a
clea enhancemen o he p in abili y o he inks, inc easing squa eness, uni o mi y, and
size accu acy while dec easing he expansion a io, excep in he case o CHIMe-NMBA
ink. The nega i e effec on he p in abili y o he addi ion o his sho -leng h c osslinking
agen can be asc ibed o he b i leness o pho ocu ed CHIMe-NMBA gels, acco ding o
he analyses o hei mechanical p ope ies. On he con a y, he long-chain and lexible
na u e o he PEG polyme in he diac yla e c osslinke led o a signi ican ly highe im-
p o emen o he p in abili y o he ink a aining squa eness and uni o mi y alues close
o 1, as well as an impo an imp o emen in size accu acy and he expansion a io. The
indings unde sco e he pi o al ole o polyme elas ici y in de e mining p in ing quali y.
The obse ed enhancemen s in squa eness, uni o mi y, and size accu acy and he educ-
ion in expansion a io a e all indica i e o he signi ican in luence ha polyme elas ici y
exe s on he o e all p in abili y o he ink. The con as ing ou comes be ween he sho -
leng h c osslinking agen NMBA, leading o b i leness, and he long-chain and lexible
PEG polyme , con ibu ing o imp o ed p in abili y, emphasize he impo ance o selec -
ing c osslinking agen s wi h app op ia e elas ic p ope ies in 3D p in ing applica ions.
Figu e 8. Op ical mic oscope images o 8
×
8 squa e sca olds o CHIMe, CHIMe-NMBA, CHIMe-
PEG, and CHIMe-AA p in ed unde 405 nm and 190 W/m2ligh .
Acco ding o Figu e 9, he addi ion o ex e nal c osslinking in CHIMe ink leads o a
clea enhancemen o he p in abili y o he inks, inc easing squa eness, uni o mi y, and
size accu acy while dec easing he expansion a io, excep in he case o CHIMe-NMBA
ink. The nega i e e ec on he p in abili y o he addi ion o his sho -leng h c osslinking
agen can be asc ibed o he b i leness o pho ocu ed CHIMe-NMBA gels, acco ding o he
analyses o hei mechanical p ope ies. On he con a y, he long-chain and lexible na u e
o he PEG polyme in he diac yla e c osslinke led o a signi ican ly highe imp o emen
o he p in abili y o he ink a aining squa eness and uni o mi y alues close o 1, as
well as an impo an imp o emen in size accu acy and he expansion a io. The indings
unde sco e he pi o al ole o polyme elas ici y in de e mining p in ing quali y. The
obse ed enhancemen s in squa eness, uni o mi y, and size accu acy and he educ ion in
expansion a io a e all indica i e o he signi ican in luence ha polyme elas ici y exe s
on he o e all p in abili y o he ink. The con as ing ou comes be ween he sho -leng h
c osslinking agen NMBA, leading o b i leness, and he long-chain and lexible PEG
polyme , con ibu ing o imp o ed p in abili y, emphasize he impo ance o selec ing
c osslinking agen s wi h app op ia e elas ic p ope ies in 3D p in ing applica ions. This
insigh is aluable o op imizing ink o mula ions and ailo ing hem o speci ic p in ing
equi emen s, ul ima ely ad ancing he quali y and pe o mance o he p in ed s uc u es.
The e ec o he ligh in ensi y was also es ed, obse ing ha , as expec ed, an in-
c ease in ligh in ensi y om 20 o 190 W/m
2
led in all cases o a clea imp o emen o
p in ing accu acy due o he highe pho ocu ing con e sion and hus c osslinking densi y
in he inal hyd ogels. Al hough he p in ing quali y o he s udied inks seems o be in lu-
enced by he ype o added c osslinking agen , showing ha NMBA de i a i es sligh ly
Gels 2024,10, 126 16 o 16
32.
Di Giuseppe, M.; Law, N.; Webb, B.; Mac ae, R.A.; Liew, L.J.; Se combe, T.B.; Dilley, R.J.; Doyle, B.J. Mechanical beha iou o
algina e-gela in hyd ogels o 3D biop in ing. J. Mech. Beha . Biomed. Ma e . 2018,79, 150–157. [C ossRe ] [PubMed]
33.
Ouyang, L.; Yao, R.; Zhao, Y.; Sun, W. E ec o bioink p ope ies on p in abili y and cell iabili y o 3D bioplo ing o emb yonic
s em cells. Bio ab ica ion 2016,8, 035020. [C ossRe ] [PubMed]
Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
