Ci a ion: An ezana, P.E.; Municoy, S.;
O i e, G.; Desimone, M.F. Design o a
New 3D Gela in—Algina e Sca old
Loaded wi h Cannabis sa i a Oil.
Polyme s 2022,14, 4506.
h ps://doi.o g/10.3390/
polym14214506
Academic Edi o s: Chao Xu and
Mu a Gu endi en
Recei ed: 14 Sep embe 2022
Accep ed: 21 Oc obe 2022
Published: 25 Oc obe 2022
Publishe ’s No e: MDPI s ays neu al
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Licensee MDPI, Basel, Swi ze land.
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polyme s
A icle
Design o a New 3D Gela in—Algina e Sca old Loaded wi h
Cannabis sa i a Oil
Pablo Edmundo An ezana 1,2, So ía Municoy 1, Go ka O i e 2,3,4,5,6 and Ma ín Fede ico Desimone 1,*
1Facul ad de Fa macia y Bioquímica, Ins i u o de Química y Me abolismo del Fá maco (IQUIMEFA),
Uni e sidad de Buenos Ai es, Consejo Nacional de In es igaciones Cien í icas y Técnicas (CONICET),
Junín 956, Buenos Ai es 1113, A gen ina
2NanoBioCel Resea ch G oup, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU),
01006 Vi o ia-Gas eiz, Spain
3Bioa aba, NanoBioCel Resea ch G oup, 01009 Vi o ia-Gas eiz, Spain
4Biomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN),
Ins i u e o Heal h Ca los III, A Mon o e de Lemos 3-5, 28029 Mad id, Spain
5Uni e si y Ins i u e o Regene a i e Medicine and O al Implan ology-UIRMI (UPV/EHU-Fundación
Edua do Ani ua), 01007 Vi o ia-Gas eiz, Spain
6Singapo e Eye Resea ch Ins i u e, The Academia, 20 College Road, Disco e y Towe ,
Singapo e 169856, Singapo e
*Co espondence: desimone@ yb.uba.a
Abs ac :
The e is an inc easing medical need o he de elopmen o new ma e ials ha could
eplace damaged o gans, imp o e healing o c i ical wounds o p o ide he en i onmen equi ed o
he o ma ion o a new heal hy issue. The h ee-dimensional (3D) p in ing app oach has eme ged
o o e come se e al o he majo de iciencies o issue enginee ing. The use o Cannabis sa i a as a
he apy o some diseases has sp ead h oughou he wo ld hanks o i s bene i s o pa ien s. In his
wo k, we de eloped a bioink made wi h gela in and algina e ha was able o be p in ed using an
ex usion 3D biop in e . The sca olds ob ained we e lyophilized, cha ac e ized and he swelling was
assessed. In addi ion, he sca olds we e loaded wi h Cannabis sa i a oil ex ac . The p esence o he
ex ac p o ided an imic obial and an ioxidan ac i i y o he 3D sca olds. Al oge he , ou esul s
sugges ha he new biocompa ible ma e ial p in ed wi h 3D echnology and wi h he addi ion
o Cannabis sa i a oil could become an a ac i e al e na i e o common ea men s o so - issue
in ec ions and wound epai .
Keywo ds: biop in ing; sca old; gela in; algina e; Cannabis sa i a
1. In oduc ion
The e is an inc easing medical need o he de elopmen o new ma e ials ha could
eplace damaged o gans, imp o e healing o c i ical wounds o p o ide he en i onmen
equi ed o he o ma ion o a new heal hy issue [
1
,
2
]. In addi ion o he ac ha cu -
en ea men s mean excessi e cos s [
3
], ch onic o non-healing wounds a e ela ed o
signi ican mo bidi y and mo ali y a es [
4
]. In his sense, he need o de elop al e na i e
echnologies wi h mo e e icien ea men s o issue epai emains a global conce n.
The h ee-dimensional (3D) p in ing app oach has eme ged o o e come se e al o he
majo de iciencies o issue enginee ing [
5
]. Using a digi al model, 3D-p in ing echnology
c ea es new biocompa ible ma e ials wi h mo e s able and pe sonalized h ee-dimensional
s uc u es wi h be e mimicking beha io o human o gans [
6
–
8
]. Compa ed o he con en-
ional skin egene a ion echniques, 3D-p in ed de mal subs i u es o e mo e au oma ion
o clinical applica ions and highe p ecision o adding li ing cells and g ow h ac o s [
9
].
To add ess his, 3D-p in ing app oaches use biop in able ma e ials, known as bioinks,
o manu ac u e 3D issue s uc u es wi h compu e -designed geome ies. Bioinks a e a
c i ical componen du ing he a i icial issue ab ica ion, and hei selec ion depends on
Polyme s 2022,14, 4506. h ps://doi.o g/10.3390/polym14214506 h ps://www.mdpi.com/jou nal/polyme s
Polyme s 2022,14, 4506 2 o 17
he speci ic applica ion [
10
,
11
]. Al hough hey can be made o syn he ic polyme s [
12
],
mos bioinks a e p epa ed wi h na u ally-de i ed bioma e ials [
13
–
15
] as hey p o ide
a sui able en i onmen o cell adhesion, p oli e a ion, di e en ia ion and he o ma ion
o a new issue. Fo example, algina e and gela in a e some o he mos equen ly used
bioinks o 3D p in ing o issue models due o hei high biocompa ibili y, low cos , good
biodeg adabili y, abundance and a ailabili y [
16
–
18
]. Gela in (GEL) is de i ed om an
acidic o basic pa ial hyd olysis o collagen, which is a majo p o ein o he na u al ex acel-
lula ma ix [
19
,
20
]. Gela in is especially a ac i e as a bioink since i has good p in abili y,
inc eases cell adhesion and can be easily deg aded by p o eases. Howe e , due o i s em-
pe a u e sensi i i y and low mechanical s eng h, gela in is gene ally combined wi h o he
bioma e ials [
21
–
23
] o imp o e mechanical p ope ies, enhance p in abili y and inc ease
cell a achmen . Algina e (ALG) is a linea anionic polysaccha ide wi h good gela ion p op-
e ies and can be easily solidi ied wi h di alen ions (like Ca
2+
, Mg
2+
and Ba
2+
) wi hou
he use o addi ional chemical c oss-linke s [
24
,
25
]. In pa icula , algina e is ex ensi ely
used o ab ica e 3D s uc u es o issue egene a ion, owing o i s biocompa ibili y, low
oxici y, biodeg adabili y, low p ice and simple gela ion p ocesses [
26
]. Simila ly o GEL,
ALG is usually blended wi h o he polyme s o enhance i s p ope ies [
27
]. The e o e,
he mul icomponen ma e ial based on GEL–ALG is an excellen candida e om which o
ob ain a bioink o he ab ica ion o 3D p in ing sca olds wi h desi ed ea u es.
Nowadays, he need o de elop inno a i e ma e ials ha could eplace and epai
damaged issues leads o he sea ch o mul i unc ional sys ems ha combine an imic obial,
an ioxidan and an i-in lamma o y p ope ies wi h a sui able ma ix o s imula e cell
p oli e a ion. Pa icula ly, he combina ion o na u al plan ex ac s wi h di e en kinds o
d essings is an a ac i e al e na i e o wound-healing ea men s due o he syne gis ic
ac ion o hei bioac i e phy ochemicals [28–30].
Cannabis sa i a L. is one o he mos ancien plan s used by humans o medical pu -
poses [
31
]. Despi e he es ic ed used o Cannabis sa i a (CS), he medical indus y o his
annual plan has g own conside ably in he las yea s. The use o Cannabis as a he apy o
some diseases o elie o symp oms de i ed om a ious ea men s has sp ead h ough-
ou he wo ld hanks o he nume ous pha macological e ec s and bene i s o pa ien s [
32
].
CS (hemp) is an he baceous dioecious plan o he Cannabinaceae amily. I s a ac i e
pha macological p ope ies come om he bioac i e phy ocannabinoids compounds na -
u ally p esen in CS [
33
]. Fo example, he main componen s
∆
9- e ahyd ocannabinol
(THC) and cannabidiol (CBD) in e ac di e en ially wi h he endocannabinoid sys em in
humans, ha ing psycho opic and non-psycho opic ac i i y, espec i ely [34]. They ha e
been p esc ibed o deal wi h dep ession, sleep diso de s and o he ea men o se e e
diseases like epilepsy, cance and mul iple scle osis. Mo eo e , ecen wo ks demons a ed
he he apeu ic po en ial o CS o imp o e human skin wound healing [
35
–
37
]. The e
is e idence ha cannabinoids can egula e cellula and molecula pa hways in ol ed in
wound es o a ion, ha e an i-in lamma o y p ope ies, educe ib osis and p oli e a ion
o hype p oli e a i e ke a inocy es and accele a e inju y closu e [
38
]. In addi ion o his,
nume ous epo s ha e demons a ed he excellen an ibac e ial ac i i y o CS, p incipally
agains G am-posi i e bac e ia [
39
,
40
]. In his sense, al hough GEL–ALG 3D-p in ed sca -
olds ha e been desc ibed o di e en applica ions [
41
], o ou knowledge his is he i s
ime ha he inclusion o Cannabis sa i a oil ex ac has been used in a 3D-p in ed sca old
o skin wound healing.
In his wo k, a GEL–ALG bioink wi h good p in abili y p ope ies was p epa ed o
ab ica e a idimensional p in ed sca old o wound healing. The biocompa ible ma e ial
was loaded wi h Cannabis sa i a oil ex ac o ob ain a 3D sca old wi h an imic obial
and an ioxidan ac i i y. Al oge he , hese esul s con i m he de elopmen o a new
biocompa ible ma e ial p in ed wi h 3D echnology and unc ionalized wi h Cannabis sa i a
oil, leading o a p omising al e na i e o common ea men s o wound in ec ions and o
wound-healing applica ions.
Polyme s 2022,14, 4506 3 o 17
2. Ma e ials and Me hods
2.1. Syn hesis o Gela in and Algina e Bioink
Type A gela in om po cine skin (Sigma Ald ich, S . Louis, MO, USA) and low-
iscosi y sodium algina e de i ed om b own algae (Sigma Ald ich) we e used. Di e en
p epa a ion p ocedu es we e e alua ed, such as he empe a u e and he s i ing ime, un il
he bes se -up condi ions we e achie ed. The bioink was made by mixing gela in 0.7 g and
algina e 0.3 g in 10 mL o PBS.
2.2. Biop in ing Pa ame e s
The bioink was p in ed using a Bio X ex usion-based 3D biop in e om Cellink
(San Ca los, CA, USA). Th ough a 27 G conical nozzle, 20 mm
×
20 mm squa e g id-
shaped sca olds we e p in ed. Hea OSTM so wa e was used o p in ing; and he model
was designed using he OBJ Viewe designing p og am. The bioinks we e p in ed on
he 3D biop in e , in which he op imal p in ing pa ame e s we e de e mined and we e
subsequen ly used wi h all he p in s (170 kPa p essu e and 27 ◦C empe a u e).
2.3. Rheological P ope ies
Fi s , ampli ude sweeps we e pe o med o de e mine he linea iscoelas ic ange.
The elas ic and he iscous moduli, G’ and G”, espec i ely, we e de e mined by small-
ampli ude oscilla o y shea low expe imen s, using a heome e AR2000 (TA ins umen s,
New Cas le, DE, USA). Fo his, pa allel pla es wi h a diame e o 20 mm we e used and
he esponse o he ma e ial in a 0.001–10 Hz ange o equency and ension 1% we e
e alua ed a 27 ◦C
2.4. C oss-Linking o he GEL–ALG Sca olds
The sca olds ob ained by biop in ing we e imme sed in a CaCl
2
solu ion o ca y ou
he c oss-linking p ocedu e. Di e en concen a ions o CaCl
2
we e e alua ed un il he
op imum o 100 mM was achie ed.
2.5. GEL–ALG Sca olds Cha ac e iza ion
2.5.1. GEL–ALG Sca olds Lyophiliza ion
A e c oss-linking and gela ion, he sca olds we e subjec ed o a lyophiliza ion
p ocess o 42 h using a Tels a eeze-d ye (Lyobe a, Ta asa, Spain). Fo his, he ollowing
d ying cycle was p og ammed: eezing o 3 h, p ima y d ying o 12 h and seconda y
d ying o 24 h. The p essu e was adjus ed o 0.2 mBa du ing p ima y d ying. Table 1
summa izes he exac p ocess pa ame e s.
Table 1. Lyophiliza ion Pa ame e s.
F eezing-D ying S ep Tempe a u e (◦C) Time (h) P essu e (mBa )
F eeze −50 3 -
Vacuum chambe 0.2
P ima y d ying −50
20
5
7
0.2
0.2
Seconda y d ying 20 24 -
2.5.2. GEL–ALG Sca olds Swelling
The sca olds we e d y-weighed, hen imp egna ed wi h PBS and inally, a e de e -
mining ha all he PBS was abso bed, hey we e weighed again. In o de o quan i y he
abso p ion capaci y o he gel, he ollowing calcula ion was pe o med:
Swelling (%) = Wwe −Wd ied
Wd ied
×100
Polyme s 2022,14, 4506 4 o 17
2.5.3. GEL–ALG Sca olds Scanning Elec on Mic oscopy (SEM)
Fo mo phological cha ac e iza ion, samples we e lyophilized as indica ed in
Sec ion 2.5.1
.
C oss-sec ions o he lyophilized ilm samples we e hen cu using a cold scalpel. Finally, he sam-
ples we e examined wi h a Jeol JSM-5400 scanning elec on mic oscope (JEOL, Tokyo, Japan).
2.5.4. Cy o oxici y Expe imen s
3T3 ib oblas s we e used o s udy he biocompa ibili y o he sca olds. The cells
we e cul u ed in a humidi ied chambe (95% ai ; 5% CO
2
) a 37
◦
C in Dulbecco’s Modi ied
Eagle’s Medium (DMEM) and supplemen ed wi h 10% e al cal se um and 1% penicillin–
s ep omycin. Once in con luence, cells we e ypsinized and coun ed by a Neubaue
came a. On op o each sca old, 1
×
10
4
cells we e seeded wi h 1000
µ
L o DMEM and
we e incuba ed a 37
◦
C o 24 and 48 h. The colo ime ic 3-(4,5-dime hyl- hiazol-2-yl)-2,5-
diphenyl- e azolium b omide (MTT) assay was used o e alua e he GEL–ALG sca old
biocompa ibili y. A e 24 o 48 h, we emo ed he medium, added 0.5 mL o MTT solu ion
(5 mg/mL) and incuba ed o 3 h a 37
◦
C. Then he MTT solu ion was disca ded and 0.5 mL
o absolu e e hanol was added a e washing he sca olds wi h PBS 1X. The abso bance alues
we e measu ed a 570 nm and esul s a e exp essed as mean
±
SD om iplica e expe imen s.
2.6. Addi ion o Cannabis sa i a Oil o GEL–ALG Sca old (GEL–ALG–CS Sca old)
To p epa e he GEL–ALG–CS sca olds, 400
µ
L o Cannabis sa i a oil ex ac we e
added o he GEL–ALG sca olds o 24 h. Cannabis sa i a oil possesses: 0.066 mg/mL
cannabidiol (CBD), 0.036 mg/mL cannabidiol acid (CBDA), 0.030 mg/mL cannabinol
(CBN), 1.267 mg/mL
∆
9- e ahyd ocannabinol (THC) and 0.226 e ahid ocannabinol
acid (THCA), de e mined by HPLC-UV (high pe o mance liquid ch oma og aphy wi h
spec oscopy UV de ec o ). Fo HPLC analysis, samples we e dilu ed and ex ac ed wi h
me hanol (HPLC-quali y). S anda d solu ions o HPLC-quali y cannabinoids (Ce illian )
we e used and ibup o en SCBT was used as in e nal s anda d. Ch oma og ams we e
ob ained using an Agilen Technologies, CA, USA 1200 Se ies HPLC wi h a Diode A ay
De ec o and a C18 column. The mobile phase was composed o ace oni ile (HPLC quali y)
and o ma e bu e solu ion (Biopack). This me hod, HPLC-UV, was used o de e mine he
elease o he di e en componen s o he Cannabis sa i a oil ex ac du ing ime.
2.6.1. Fou ie T ans o m In a ed (FTIR) Analysis
FTIR spec a o Cannabis sa i a oil ex ac , GEL–ALG sca old and GEL–ALG–CS
sca old we e ob ained o e he ange o 4000–500 cm
−1
, using a FTIR-Raman Nicole iS 50
(The mo Scien i ic, Wal ham, MA, USA). Samples was d ied unde a ni ogen low and he
powde was hen placed on he a enua ed o al e lec ion accesso y o he spec ome e [
42
].
2.6.2. Sca old Enzyma ic Deg ada ion
We assess he sca olds’ deg ada ion by collagenase diges ion, incuba ing GEL–ALG
sca old and GEL–ALG–CS sca olds wi h collagenase enzyme solu ion in PBS a 37
◦
C
(1 mL o a 10 U/mL solu ion o ype I collagenase Gibco
®
, 260 U/mg). Sca old’s weigh s
we e measu ed a di e en ime in e als [43].
2.6.3. An ioxidan Capaci y
To s udy he an ioxidan ac i i y o he sca olds, DPPH colo ime ic assay was pe -
o med [
29
]. This me hod is based on he measu emen o he sca enging ac i i y o he
2,2-diphenyl-1-pic ylhyd azyl ee adical (DPPH
•
). To s udy he an ioxidan ac i i y o
Cannabis sa i a, 3 mL o a me hanolic solu ion o DPPH•(25 mg/L) we e added o 200 µL
o he CS ex ac . Fo GEL–ALG and GEL–ALG–CS, he same olume o DPPH
•
(25 mg/L)
was added o he sca olds and incuba ed o 5 min o 10 min a oom empe a u e. In addi-
ion, wo GEL–ALG–CS sca olds wi h di e en weigh s we e e alua ed. The abso bances
Polyme s 2022,14, 4506 5 o 17
we e e alua ed a 517 nm. The an ioxidan capaci y o CS, GEL–ALG and GEL–ALG–CS
was calcula ed as pe cen age o inhibi ion wi h he ollowing equa ion:
%inhibi ion = [1 −(Abssample/AbsDPPH solu ion)] ×100
2.6.4. An imic obial Ac i i y
We e alua ed he bac e icidal ac i i y o GEL–ALG sca old and GEL–ALG–CS sca old
on G am-posi i e (S aphylococcus au eus) and G am-nega i e (Esche ichia coli) bac e ia. To
each he op imal g ow h condi ions o S. au eus (ATCC 29213) and E. coli (ATCC 9637),
hey we e incuba ed o e nigh a 37
◦
C in yp ic soy aga (TSA) medium (enzyma ic diges
o casein 15 g/L, enzyma ic diges o soybean 5 g/L, NaCl 5/L, aga 15 g/L). Then, hey
we e dilu ed o ob ain 1
×
10
6
Colony Fo ming Uni s (CFU) pe mL. The an imic obial
ac i i y was e alua ed using wo me hods: he solid and he dilu ion me hod. Fo he
disk di usion me hod, 20
µ
L o a bac e ial suspension 1:1000 was sp ead on an aga Pe i
dish. Then, GEL–ALG and GEL–ALG–CS sca olds we e placed on he aga su ace and
incuba ed a 37
◦
C. The inhibi o y ac ion o he sca olds was de e mined a e 24 h by
measu ing he diame e o he bac e ia- ee a eas su ounding he sca olds.
On he o he hand, o s udy he an imic obial capaci y o he sca olds by he dilu ion
me hod, 100
µ
L o he bac e ial suspension 1:1000 was mixed wi h Cannabis sa i a oil ex ac
and GEL–ALG sca old and GEL–ALG–CS sca olds wi h 900
µ
L o Lu ia Be ani (LB)
medium (yeas ex ac , 5 g/L; NaCl, 10 g/L; and yp one, 10 g/L). The samples we e
incuba ed a 37
◦
C o 24 h. A e ha , se ial dilu ions om he supe na an s we e made in
PBS. Aliquo s o 20
µ
L o he dilu ions we e seeded on aga Pe i dishes and main ained
a 37
◦
C o 12 h. CFU we e de e mined by manually coun ing he colonies on he pla e.
Resul s a e exp essed as mean ±SD om iplica e expe imen s.
2.6.5. Biocompa ibili y
Biocompa ibili y was e alua ed in 3T3 ib oblas s, as p e iously desc ibed in
Sec ion 2.5.4, using MTT assay.
2.7. S e iliza ion
All he sca olds we e s e ilized o 30 min using UV-C ligh , since i has ge micidal
ac i i y [
44
]. The s e iliza ion was con i med using an adap ed me hod om he Eu opean
Pha macopoeia: in b ie , he s e ilized sca old was incuba ed in LB a 37
◦
C o 7 days and
cul i a ed in a pe i dish. No g ow h was obse ed a e he 7 days e alua ed.
2.8. S a is ical Analysis
S a is ical analysis was pe o med using he s a is ical so wa e G aphPad P ism 5.0
so wa e (G aphPad So wa e, La Jolla, CA, USA). Da a we e exp essed as mean
±
SD o
iplica e expe imen s. The di e ences we e analyzed using one-way ANOVA, ollowed by
he Tuckey’s pos - es ; p< 0.05 was conside ed signi ican .
3. Resul s and Discussion
3.1. Syn hesis and Cha ac e iza ion o Gela in and Algina e Bioink
In o de o de elop a good, p in able bioink, i is essen ial o de e mine he bes
p epa a ion p ocess. In his sense, we p obed h ee di e en p ocedu es by mixing he
gela in and algina e in h ee di e en condi ions, as is shown in Table 2.
Table 2. Di e en condi ions du ing ink p epa a ion.
Condi ion 1 Condi ion 2 Condi ion 3
Mix gela in + algina e, hen add
PBS and inally s i a
60 ◦C o 2 h.
Mix gela in + algina e, hen add
PBS and inally s i a oom
empe a u e o 2 h
Fi s add gela in o PBS, hen add
algina e in PBS, mix hese
solu ions and inally s i a
60 ◦C o 2 h.
Polyme s 2022,14, 4506 6 o 17
Du ing he biop in ing p ocess an ex usion me hod was used, as desc ibed in he
Ma e ials and Me hods sec ion. We used pneuma ic p essu e o ex ude he ink om
he needle in an unin e up ed line [
45
]. A e he biop in ing p ocess, we ound ha
inks p epa ed unde Condi ions 2 and 3 we e no able o be p in ed. On he o he hand,
Condi ion 1 allowed us o p epa e an ink ha was p in ed success ully (Figu e 1).
Polyme s 2022, 14, 4506 6 o 16
3. Resul s and Discussion
3.1. Syn hesis and Cha ac e iza ion o Gela in and Algina e Bioink
In o de o de elop a good, p in able bioink, i is essen ial o de e mine he bes p ep-
a a ion p ocess. In his sense, we p obed h ee di e en p ocedu es by mixing he gela in
and algina e in h ee di e en condi ions, as is shown in Table 2.
Table 2. Di e en condi ions du ing ink p epa a ion.
Condi ion 1 Condi ion 2 Condi ion 3
Mix gela in + algina e, hen add
PBS and inally s i a 60 °C o
2 h.
Mix gela in + algina e, hen
add PBS and inally s i a
oom empe a u e o 2 h
Fi s add gela in o PBS,
hen add algina e in PBS,
mix hese solu ions and i-
nally s i a 60 °C o 2 h.
Du ing he biop in ing p ocess an ex usion me hod was used, as desc ibed in he
Ma e ials and Me hods sec ion. We used pneuma ic p essu e o ex ude he ink om he
needle in an unin e up ed line [45]. A e he biop in ing p ocess, we ound ha inks
p epa ed unde Condi ions 2 and 3 we e no able o be p in ed. On he o he hand, Con-
di ion 1 allowed us o p epa e an ink ha was p in ed success ully (Figu e 1).
Figu e 1. GEL–ALG sca olds p in ed unde he h ee condi ions.
The e o e, he i s s ep o p epa e he bioink was o mix gela in and algina e, hen o
add he PBS and inally o shake a 60 °C o 2 h. In he case o Condi ion 2, i is possible
ha he lack o empe a u e a ec ed he o ma ion o a homogeneous mix u e. This could
be p obably due o he ac ha gela in is sensi i e o empe a u e and i s abili y o gene -
a e s uc u es is de e mined by empe a u e [46]. On he o he hand, i is possible ha in
Condi ion 3 he p oblem was in he dissolu ion o he algina e. This is due o he ac ha
algina e equi es immedia e and cons an agi a ion om he beginning o i s dissolu ion
in o de o ob ain a homogeneous consis ency and make an accu a e p in ing [47,48].
We e alua ed he mechanical p ope ies and s abili y o GEL–ALG bioink by s udy-
ing i s heological beha io a 25° C. This es de e mines whe he he bioink has a p e-
dominan ly elas ic o iscous beha io . G’ co esponds o he elas ic componen , while G”
co esponds o he iscous one. The ela ionship o he wo modules p o ides in o ma ion
on he cha ac e is ics o he sample. I is widely known ha any ype o ma e ial has a
solid componen and a liquid componen . Depending on he p edominan componen in
each case, we can classi y he ma e ials as iscoelas ic solids o iscoelas ic liquids. This
s ep is c ucial o he success o clinical applica ions while de eloping a new bioma e ial.
As i is shown in Figu e 2A, he ink possesses a gel-like beha io , since he alues o G’
elas ic componen s exceed he alues o G” iscous componen s. Figu e 2B displays a
Figu e 1. GEL–ALG sca olds p in ed unde he h ee condi ions.
The e o e, he i s s ep o p epa e he bioink was o mix gela in and algina e, hen o
add he PBS and inally o shake a 60
◦
C o 2 h. In he case o Condi ion 2, i is possible
ha he lack o empe a u e a ec ed he o ma ion o a homogeneous mix u e. This could
be p obably due o he ac ha gela in is sensi i e o empe a u e and i s abili y o gene a e
s uc u es is de e mined by empe a u e [
46
]. On he o he hand, i is possible ha in
Condi ion 3 he p oblem was in he dissolu ion o he algina e. This is due o he ac ha
algina e equi es immedia e and cons an agi a ion om he beginning o i s dissolu ion in
o de o ob ain a homogeneous consis ency and make an accu a e p in ing [47,48].
We e alua ed he mechanical p ope ies and s abili y o GEL–ALG bioink by s udying
i s heological beha io a 25
◦
C. This es de e mines whe he he bioink has a p edom-
inan ly elas ic o iscous beha io . G’ co esponds o he elas ic componen , while G”
co esponds o he iscous one. The ela ionship o he wo modules p o ides in o ma ion
on he cha ac e is ics o he sample. I is widely known ha any ype o ma e ial has a
solid componen and a liquid componen . Depending on he p edominan componen in
each case, we can classi y he ma e ials as iscoelas ic solids o iscoelas ic liquids. This
s ep is c ucial o he success o clinical applica ions while de eloping a new bioma e ial.
As i is shown in Figu e 2A, he ink possesses a gel-like beha io , since he alues o G’
elas ic componen s exceed he alues o G” iscous componen s. Figu e 2B displays a
pho og aph o he GEL–ALG sca old a e p in ing, showing ha he bioink was p in ed
wi h p ecision.
Polyme s 2022,14, 4506 7 o 17
Polyme s 2022, 14, 4506 7 o 16
pho og aph o he GEL–ALG sca old a e p in ing, showing ha he bioink was p in ed
wi h p ecision.
Figu e 2. (A) Va ia ion o he elas ic (G’, ed c osses) and iscous modulus (G”, blue iangles) wi h
equency o GEL–ALG ink. (B) GEL–ALG sca old pho og aph a e p in ing.
Once he sca olds had been p in ed, we p oceeded wi h he c oss-linking. The bi-
op in ed GEL–ALG sca olds we e c oss-linked wi h 100 mM CaCl
2
(Figu e 3). The c oss-
linking allows an imp o emen in he s uc u e o he sca old, leading o a dec ease in i s
deg adabili y and an inc ease in i s s abili y [49]. In addi ion, he use o calcium sal , which
is an economical compound, is a o able when conside ing p oduc ion on la ge scales.
Figu e 3. GEL–ALG sca olds pho og aph a e c oss-linking wi h CaCl
2
.
A e achie ing a p in able bioink, we decided o lyophilize he GEL–ALG sca olds.
A e his p ocedu e, i was in e es ing o obse e ha he sca olds main ained hei s uc-
u e and dimensions (Figu e 4A). As a esul o his, i can be concluded ha he sca olds
could be p in ed, lyophilized and hen, a he ime o use, ehyd a ed and/o imp egna ed
wi h he apeu ic molecules. This would allow a mul i unc ional ac ion o i s de mal ap-
plica ion.
Ano he impo an ea u e o be cha ac e ized is he swelling capaci y o he sca old.
In his sense, swelling is impo an o a biomedical ma e ial because i is ela ed o he
exchange o subs ances as well as o he p ope ies, such as mechanical p ope ies o lex-
ibili y [50]. We ound ha GEL–ALG sca olds each he maximum PBS up ake a 6 h,
emaining cons an o mo e han 50 h. In addi ion, he GEL–ALG sca olds eached 800%
o swelling (Figu e 4B).
The nex s ep was o s udy he s uc u e o he sca olds h ough SEM. The use o his
mic oscopy is in e es ing, since i is a echnique ha allows a wide a ie y o ma e ials o
be cha ac e ized bo h a mac o- and mic oscale; among hese can be men ioned nano-
s uc u ed ma e ials, me al alloys, polyme s, mine als, ibe s, hin ilms and bioma e ials
Figu e 2.
(
A
) Va ia ion o he elas ic (G’, ed c osses) and iscous modulus (G”, blue iangles) wi h
equency o GEL–ALG ink. (B) GEL–ALG sca old pho og aph a e p in ing.
Once he sca olds had been p in ed, we p oceeded wi h he c oss-linking. The biop in ed
GEL–ALG sca olds we e c oss-linked wi h 100 mM CaCl
2
(Figu e 3). The c oss-linking allows
an imp o emen in he s uc u e o he sca old, leading o a dec ease in i s deg adabili y and
an inc ease in i s s abili y [
49
]. In addi ion, he use o calcium sal , which is an economical
compound, is a o able when conside ing p oduc ion on la ge scales.
Polyme s 2022, 14, 4506 7 o 16
pho og aph o he GEL–ALG sca old a e p in ing, showing ha he bioink was p in ed
wi h p ecision.
Figu e 2. (A) Va ia ion o he elas ic (G’, ed c osses) and iscous modulus (G”, blue iangles) wi h
equency o GEL–ALG ink. (B) GEL–ALG sca old pho og aph a e p in ing.
Once he sca olds had been p in ed, we p oceeded wi h he c oss-linking. The bi-
op in ed GEL–ALG sca olds we e c oss-linked wi h 100 mM CaCl
2
(Figu e 3). The c oss-
linking allows an imp o emen in he s uc u e o he sca old, leading o a dec ease in i s
deg adabili y and an inc ease in i s s abili y [49]. In addi ion, he use o calcium sal , which
is an economical compound, is a o able when conside ing p oduc ion on la ge scales.
Figu e 3. GEL–ALG sca olds pho og aph a e c oss-linking wi h CaCl
2
.
A e achie ing a p in able bioink, we decided o lyophilize he GEL–ALG sca olds.
A e his p ocedu e, i was in e es ing o obse e ha he sca olds main ained hei s uc-
u e and dimensions (Figu e 4A). As a esul o his, i can be concluded ha he sca olds
could be p in ed, lyophilized and hen, a he ime o use, ehyd a ed and/o imp egna ed
wi h he apeu ic molecules. This would allow a mul i unc ional ac ion o i s de mal ap-
plica ion.
Ano he impo an ea u e o be cha ac e ized is he swelling capaci y o he sca old.
In his sense, swelling is impo an o a biomedical ma e ial because i is ela ed o he
exchange o subs ances as well as o he p ope ies, such as mechanical p ope ies o lex-
ibili y [50]. We ound ha GEL–ALG sca olds each he maximum PBS up ake a 6 h,
emaining cons an o mo e han 50 h. In addi ion, he GEL–ALG sca olds eached 800%
o swelling (Figu e 4B).
The nex s ep was o s udy he s uc u e o he sca olds h ough SEM. The use o his
mic oscopy is in e es ing, since i is a echnique ha allows a wide a ie y o ma e ials o
be cha ac e ized bo h a mac o- and mic oscale; among hese can be men ioned nano-
s uc u ed ma e ials, me al alloys, polyme s, mine als, ibe s, hin ilms and bioma e ials
Figu e 3. GEL–ALG sca olds pho og aph a e c oss-linking wi h CaCl2.
A e achie ing a p in able bioink, we decided o lyophilize he GEL–ALG sca olds.
A e his p ocedu e, i was in e es ing o obse e ha he sca olds main ained hei
s uc u e and dimensions (Figu e 4A). As a esul o his, i can be concluded ha he
sca olds could be p in ed, lyophilized and hen, a he ime o use, ehyd a ed and/o
imp egna ed wi h he apeu ic molecules. This would allow a mul i unc ional ac ion o i s
de mal applica ion.
Polyme s 2022,14, 4506 8 o 17
Polyme s 2022, 14, 4506 8 o 16
[51]. I is essen ial o know he in e nal and ex e nal s uc u e o he sca olds, since his
has been ecognized as an impo an ac o in de ining cell beha io [52]. When analyzing
sca olds by SEM, a compac and smoo h su ace was obse ed (Figu e 4C) whose po os-
i y could be app ecia ed when making a c oss sec ion (Figu e 4D). The size o hese po es
was de e mined, con i ming ha i is a highly po ous ma e ial wi h an a e age po e size
o 15.0 ± 2.3 µm.
Figu e 4. (A) Lyophilized GEL–ALG sca old pho og aph. (B) GEL–ALG sca old swelling de e mi-
na ion. Values ep esen mean ± SD. (C) Scanning Elec on Mic oscopy (SEM) o GEL–ALG sca old.
(D) Close-up o he po ous a ea obse ed in image (C).
In his sense, he main unc ion o a sca old based on biodeg adable polyme s is ha
i mus be capable o suppo ing he adhesion, p oli e a ion and di e en ia ion o cells
un il he egene a ion o issues a an inju ed si e. Fo his eason, he po osi y o he sca -
olds is impo an , since some po e sizes can imp o e cell ac i i y. The op imal size and
geome y a e highly dependen on he speci ic cell ype g owing a he inju ed si es [53].
S udies in cells ha e shown ha ib oblas s ha e a b idging mechanism ha allows hem
o ex end hemsel es o ill po es o di e en sizes, e en he la ges ones ( om 5 o 90
mm). On he o he hand, since endo helial cells do no ha e his b idging sys em, hey
p e e po es wi h sizes close o hei own dimensions [54]. Thus, he po osi y could se e
as an ideal s uc u e o allow coloniza ion by cells h ough in il a ion and cell g ow h. I
is wo h men ioning ha a high po osi y also p o ides a a o able s uc u e o allow he
inco po a ion o di e en he apeu ic molecules. The e o e, hese sca olds a e ex emely
in e es ing o hei applica ion in u u e pe spec i es.
I is widely epo ed ha bo h gela in and algina e a e biocompa ible and used o
a ious biomedical applica ions[55,56]. Gi en ha 3D-p in ed GEL–ALG sca old could
be applied o p omo e would healing, i s cy o oxici y was also e alua ed. He ein, a ib o-
blas cell line was chosen as a model cell due o i s main ole in skin wound epa a ion.
Fo his, a 3T3 ib oblas cell cul u e was incuba ed in DMEM in he p esence o he sca -
olds, a 37 °C in a humidi y incuba o . A e 24 h, cell iabili y was de e mined by he in
Figu e 4.
(
A
) Lyophilized GEL–ALG sca old pho og aph. (
B
) GEL–ALG sca old swelling de e mi-
na ion. Values ep esen mean
±
SD. (
C
) Scanning Elec on Mic oscopy (SEM) o GEL–ALG sca old.
(D) Close-up o he po ous a ea obse ed in image (C).
Ano he impo an ea u e o be cha ac e ized is he swelling capaci y o he sca old.
In his sense, swelling is impo an o a biomedical ma e ial because i is ela ed o he
exchange o subs ances as well as o he p ope ies, such as mechanical p ope ies o
lexibili y [
50
]. We ound ha GEL–ALG sca olds each he maximum PBS up ake a 6 h,
emaining cons an o mo e han 50 h. In addi ion, he GEL–ALG sca olds eached 800%
o swelling (Figu e 4B).
The nex s ep was o s udy he s uc u e o he sca olds h ough SEM. The use o
his mic oscopy is in e es ing, since i is a echnique ha allows a wide a ie y o ma e-
ials o be cha ac e ized bo h a mac o- and mic oscale; among hese can be men ioned
nano-s uc u ed ma e ials, me al alloys, polyme s, mine als, ibe s, hin ilms and bioma e-
ials [
51
]. I is essen ial o know he in e nal and ex e nal s uc u e o he sca olds, since his
has been ecognized as an impo an ac o in de ining cell beha io [
52
]. When analyzing
sca olds by SEM, a compac and smoo h su ace was obse ed (Figu e 4C) whose po osi y
could be app ecia ed when making a c oss sec ion (Figu e 4D). The size o hese po es was
de e mined, con i ming ha i is a highly po ous ma e ial wi h an a e age po e size o
15.0 ±2.3 µm.
In his sense, he main unc ion o a sca old based on biodeg adable polyme s is
ha i mus be capable o suppo ing he adhesion, p oli e a ion and di e en ia ion o
cells un il he egene a ion o issues a an inju ed si e. Fo his eason, he po osi y o he
sca olds is impo an , since some po e sizes can imp o e cell ac i i y. The op imal size and
geome y a e highly dependen on he speci ic cell ype g owing a he inju ed si es [
53
].
S udies in cells ha e shown ha ib oblas s ha e a b idging mechanism ha allows hem o
ex end hemsel es o ill po es o di e en sizes, e en he la ges ones ( om 5 o 90 mm).
On he o he hand, since endo helial cells do no ha e his b idging sys em, hey p e e
po es wi h sizes close o hei own dimensions [
54
]. Thus, he po osi y could se e as
Polyme s 2022,14, 4506 9 o 17
an ideal s uc u e o allow coloniza ion by cells h ough in il a ion and cell g ow h. I
is wo h men ioning ha a high po osi y also p o ides a a o able s uc u e o allow he
inco po a ion o di e en he apeu ic molecules. The e o e, hese sca olds a e ex emely
in e es ing o hei applica ion in u u e pe spec i es.
I is widely epo ed ha bo h gela in and algina e a e biocompa ible and used o
a ious biomedical applica ions [
55
,
56
]. Gi en ha 3D-p in ed GEL–ALG sca old could be
applied o p omo e would healing, i s cy o oxici y was also e alua ed. He ein, a ib oblas
cell line was chosen as a model cell due o i s main ole in skin wound epa a ion. Fo his,
a 3T3 ib oblas cell cul u e was incuba ed in DMEM in he p esence o he sca olds, a
37
◦
C in a humidi y incuba o . A e 24 h, cell iabili y was de e mined by he
in i o
MTT
assay. As i was expec ed, when ib oblas s we e exposed o he GEL–ALG idimensional
ma e ial, a 70% cell iabili y was eached compa ed wi h he cell con ol (Figu e 5). This
demons a es he good biocompa ibili y o he combined bioink and i s po en ial use as a
bioma e ial o wound healing. In o de o e alua e i he GEL–ALG sca old a ec ed he
cell iabili y du ing ime, we s udied he cy o oxici y a 48 h. Figu e 5shows ha a e ha
ime he cells con inued g owing when exposed o he GEL–ALG sca old.
Polyme s 2022, 14, 4506 9 o 16
i o MTT assay. As i was expec ed, when ib oblas s we e exposed o he GEL–ALG
idimensional ma e ial, a 70% cell iabili y was eached compa ed wi h he cell con ol
(Figu e 5). This demons a es he good biocompa ibili y o he combined bioink and i s
po en ial use as a bioma e ial o wound healing. In o de o e alua e i he GEL–ALG
sca old a ec ed he cell iabili y du ing ime, we s udied he cy o oxici y a 48 h. Figu e
5 shows ha a e ha ime he cells con inued g owing when exposed o he GEL–ALG
sca old.
0
50
100
150
Con ol GEL-ALG sca old
24 h 48 h
*
*
Cell p oli e a ion (%)
Figu e 5. Viabili y o 3T3 ib oblas s e alua ed by he MTT es a 24 h o con ol and GEL–ALG
sca old. Viabili y a e in con ol was conside ed as 100%. Resul s a e exp essed as mean ± SD om
iplica e expe imen s. * p < 0.05.
3.2. Addi ion o Cannabis sa i a Oil o GEL–ALG Sca old (GEL–ALG–CS Sca old)
Due o he in e es ing p ope ies displayed by he GEL–ALG sca old, we decided o
add Cannabis sa i a oil o he sca old, leading o a concen a ion o 0.55 mg CBD/g in he
sca old (Figu e 6A). The aim was o ob ain a new bioma e ial wi h an ioxidan and an i-
mic obial p ope ies [36]. To con i m ha CS was success ully inco po a ed in o he sca -
old, an FTIR analysis was pe o med. Figu e 6B shows FTIR spec a o GEL–ALG sca -
olds, CS ex ac and GEL–ALG sca olds con aining CS (GEL–ALG–CS). GEL–ALG sca -
olds show cha ac e is ics peaks wi hin he mix u e ha demons a e a s ong in e molec-
ula a ac ion be ween bo h mac omolecules h ough elec os a ic in e ac ions [57]. GEL–
ALG spec um shows a b oad band a 3272 cm−1, assigned o NH and OH s e ching i-
b a ions o amides, and a peak a 1641 cm−1 ha co esponds o he coupling o C=O and
CN s e ching ib a ions o amide I o gela in [58] wi h he asymme ic s e ching ib a-
ions o COO- o algina e [59]. The peak a 1542 cm−1 is assigned o NH and CN ib a ion
o g oups in amide II o gela in [60], while he signals a 1031 and 1079 cm−1 a e a ibu ed
o ib a ion o C-O and CO-C g oups in mannu onic and gulu onic uni s o algina e [61],
espec i ely. The b oad peak obse ed a 3308 cm−1 in he FTIR spec um o CS ep esen s
he OH s e ching ib a ional band and he signals a 2925 and 2875 cm−1 co espond o
he symme ic and asymme ic s e ching ib a ions o CH alkane g oups. Fu he mo e,
he abso bance peaks a 1637 and 1049 cm−1 a e a ibu ed o C=C double-bond s e ching
and C-O s e ching o CS, espec i ely. Inco po a ion o CS in GEL–ALG sca olds was
con i med by analyzing he GEL–ALG–CS spec um. This shows he p esence o cha ac-
e is ic peaks o CS a 2924 and 2850 cm−1, which is no obse ed in he GEL–ALG spec-
um, and a mo e in ense band a 1641 and 1040 cm−1 which e idences he coupling o
algina e, gela in and CS signals.
Figu e 5.
Viabili y o 3T3 ib oblas s e alua ed by he MTT es a 24 h o con ol and GEL–ALG
sca old. Viabili y a e in con ol was conside ed as 100%. Resul s a e exp essed as mean
±
SD om
iplica e expe imen s. * p< 0.05.
3.2. Addi ion o Cannabis sa i a Oil o GEL–ALG Sca old (GEL–ALG–CS Sca old)
Due o he in e es ing p ope ies displayed by he GEL–ALG sca old, we decided o
add Cannabis sa i a oil o he sca old, leading o a concen a ion o 0.55 mg CBD/g in he
sca old (Figu e 6A). The aim was o ob ain a new bioma e ial wi h an ioxidan and an imi-
c obial p ope ies [
36
]. To con i m ha CS was success ully inco po a ed in o he sca old,
an FTIR analysis was pe o med. Figu e 6B shows FTIR spec a o GEL–ALG sca olds,
CS ex ac and GEL–ALG sca olds con aining CS (GEL–ALG–CS). GEL–ALG sca olds
show cha ac e is ics peaks wi hin he mix u e ha demons a e a s ong in e molecula
a ac ion be ween bo h mac omolecules h ough elec os a ic in e ac ions [
57
]. GEL–ALG
spec um shows a b oad band a 3272 cm
−1
, assigned o NH and OH s e ching ib a ions
o amides, and a peak a 1641 cm
−1
ha co esponds o he coupling o C=O and CN
s e ching ib a ions o amide I o gela in [
58
] wi h he asymme ic s e ching ib a ions
Polyme s 2022,14, 4506 16 o 17
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