Academic Edi o : Rossella Bedini
Recei ed: 15 Janua y 2025
Re ised: 10 Feb ua y 2025
Accep ed: 16 Feb ua y 2025
Published: 21 Feb ua y 2025
Ci a ion: Rocha, J.M.; Sousa, R.P.C.L.;
Sousa, D.; Tohidi, S.D.; Ribei o, A.;
Fanguei o, R.; Fe ei a, D.P.
Polycap olac one-Based Fib ous
Sca olds Rein o ced wi h Cellulose
Nanoc ys als o An e io C ucia e
Ligamen Repai . Appl. Sci. 2025,15,
2301. h ps://doi.o g/10.3390/
app15052301
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/
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A icle
Polycap olac one-Based Fib ous Sca olds Rein o ced wi h
Cellulose Nanoc ys als o An e io C ucia e Ligamen Repai
Joana M. Rocha 1, Rui P. C. L. Sousa 1,* , Diogo Sousa 1, Sha agh D. Tohidi 2, A u Ribei o 3,4 ,
Raul Fanguei o 1and Diana P. Fe ei a 1,*
1Cen e o Tex ile Science and Technology (2C2T), Uni e si y o Minho, 4800-056 Guima ães, Po ugal;
[email p o ec ed] (J.M.R.); [email p o ec ed] (D.S.); anguei [email p o ec ed] (R.F.)
2DTx CoLab-Digi al T ans o ma ion CoLab, Uni e si y o Minho, 4800-056 Guima ães, Po ugal;
[email p o ec ed]
3Cen e o Biological Enginee ing, Campus de Gual a , Uni e si y o Minho, 4710-057 B aga, Po ugal;
[email p o ec ed]
4Associa e Labo a o y in Bio echnology, Bioenginee ing and Mic oelec omechanical Sys ems (LABBELS),
B aga/Guima ães, Po ugal
*Co espondence: [email p o ec ed] (R.P.C.L.S.); [email p o ec ed] (D.P.F.)
Abs ac : An e io c ucia e ligamen (ACL) inju ies pose signi ican challenges, d i ing he
need o inno a i e epai s a egies. Tissue enginee ing (TE) has eme ged as a p omising
ield o ACL inju ies. We spinning is a ilamen p oduc ion echnique ha enables p ecise
con ol o e ilamen alignmen , diame e , and po osi y, making i sui able o de eloping
new sca olds o ACL inju ies. This s udy de elops ib ous sca olds using we spinning
o polycap olac one (PCL) ein o ced wi h cellulose nanoc ys als (CNC) o enhance me-
chanical p ope ies. We spinning was employed o ab ica e ib ous sca olds, u ilizing
PCL as he p ima y polyme due o i s a o able biocompa ibili y and deg adabili y. An
au oma ed collec o was de eloped and op imized, which allowed he s e ching o he PCL
ilamen s o diame e s as low as 30
µ
m. Se e al ilamen s we e explo ed and cha ac e ized
using SEM, TGA, and mechanical es s. The op imized PCL/CNC ilamen s we e used o
de elop 3D b aided s uc u es ha mimic ACL s uc u e. The combina ion o we -spun
PCL/CNC ilamen s (wi h an op imized s i ing me hod) and he b aiding p ocedu e
allowed o ully biocompa ible sca olds ha mimic bo h he s uc u e and he mechanical
p ope ies o na i e ACL. Cy o oxici y and biocompa ibili y es s showed cell iabili y
and p oli e a ion alues abo e 99% and 81%, espec i ely. These indings unde sco e he
po en ial o CNC- ein o ced PCL sca olds as p omising candida es o ACL epai , laying
he g oundwo k o u u e biomedical applica ions.
Keywo ds: an e io c ucia e ligamen ; polycap olac one; cellulose nanoc ys als; we
spinning; issue enginee ing
1. In oduc ion
The an e io c ucia e ligamen (ACL) is one o he key ligamen s ha helps s abilize
he knee join . The ACL connec s he emu o he ibia and is essen ial o s abilizing he
knee join . I s up u e is among he mos equen and debili a ing inju ies encoun e ed
in o hopedics [
1
]. Tendon and ligamen inju ies, pa icula ly hose in ol ing he ACL,
a ec millions o people globally each yea , imposing signi ican clinical and economic
bu dens [
2
]. Such inju ies a e o en associa ed wi h high-impac spo s, aging, o auma ic
e en s, leading o impai ed mobili y, ch onic pain, and diminished quali y o li e. ACL
inju ies p o oundly impac pa ien s’ ac i i y le els and quali y o li e, as eco e y o en
Appl. Sci. 2025,15, 2301 h ps://doi.o g/10.3390/app15052301
Appl. Sci. 2025,15, 2301 2 o 19
in ol es p olonged ehabili a ion wi h a ying success a es. Despi e ad ancemen s in
su gical echniques and ehabili a ion p o ocols, ou comes o en ail o achie e comple e
unc ional eco e y o p e en long- e m complica ions, such as e-inju y [
3
]. This e-
qui es he explo a ion o inno a i e app oaches ha can p o ide mo e e ec i e long- e m
solu ions [4].
Tissue enginee ing (TE) has eme ged as a p omising ield ha o e comes he limi-
a ions o adi ional ea men s by p omo ing issue egene a ion and mimicking na i e
issues. TE combines p inciples o biology, ma e ials science, and enginee ing o de elop
biomime ic sca olds capable o suppo ing cell g ow h, p oli e a ion, and di e en ia ion
while es o ing s uc u al in eg i y. The goal o TE is o de elop sca olds wi h mechanical
and biological p ope ies ha mi o hose o na u al ligamen s [
5
]. Among he a ious
ab ica ion echniques, we spinning has gained a en ion o i s abili y o p oduce h ee-
dimensional (3D) ib ous s uc u es wi h a o able p ope ies o issue egene a ion. We
spinning is a manu ac u ing p ocess ha in ol es ex uding a polyme ic o mula ion
h ough a needle in o a coagula ion ba h, whe e he polyme coagula es in o solid ibe s.
Unlike adi ional me hods, we spinning enables p ecise con ol o e ilamen alignmen ,
diame e , and po osi y, which a e c i ical o eplica ing he na i e ex acellula ma ix
(ECM) and achie ing issue in eg a ion [
6
]. In compa ison wi h elec ospinning and addi-
i e manu ac u ing, we spinning does no equi e high empe a u es o ol ages, making i
sui able o p ocessing highly biocompa ible polyme s. This me hod enables he con olled
o ma ion o con inuous ilamen s wi h unable diame e s and supe io mechanical p op-
e ies o la ge-scale p oduc ion, making i pa icula ly sui able o sca olds whe e high
ensile s eng h and con olled ibe alignmen a e c ucial [
7
]. B accini e al. demons a ed
ha compu e -aided we spinning (CAWS) can ab ica e chi osan/hyalu onic acid sca olds
wi h con olled mac opo osi y, enhancing cell adhesion and p oli e a ion, unde sco ing i s
po en ial o issue enginee ing applica ions [
8
]. These indings highligh he impo ance o
ad anced ab ica ion me hods in ailo ing sca old p ope ies o mee speci ic biological
and mechanical equi emen s.
The choice o ma e ials o sca old p oduc ion is c i ical, pa icula ly in he con ex o
sus ainabili y and biocompa ibili y [
9
]. The e has been a g owing emphasis on using bio-
compa ible and biodeg adable polyme s, d i en by en i onmen al conce ns and he need
o ma e ials ha a e compa ible wi h human issues [
10
]. Na u al and syn he ic polyme s,
including collagen, chi osan, silk ib oin, polycap olac one (PCL), and polylac ic acid (PLA),
ha e been widely in es iga ed o hei po en ial in ligamen issue enginee ing. Collagen
and silk ib oin exhibi excellen biocompa ibili y bu o en lack he necessa y mechanical
s eng h o ACL econs uc ion [
11
,
12
]. PLA-based sca olds o e unable biodeg adabili y,
ye hei b i le na u e can limi long- e m mechanical s abili y [
13
,
14
]. An ideal ma e ial
o ACL epai mus balance biocompa ibili y and mechanical s eng h, ensu ing bo h
cellula in eg a ion and su icien load-bea ing capaci y. In e ms o biodeg adabili y, i
should deg ade a a con olled a e o p o ide s uc u al suppo h oughou he healing
p ocess while g adually being eplaced by egene a ed issue. Polycap olac one (PCL) has
been iden i ied as one o he mos p omising syn he ic polyme s o issue enginee ing
applica ions, especially o long- e m use, due o i s slow deg ada ion a e, high mechanical
s eng h, and ease o p ocessing [
15
–
17
]. PCL’s abili y o main ain mechanical s abili y
o e ex ended pe iods makes i pa icula ly well-sui ed o applica ions whe e p olonged
suppo is necessa y, such as in ACL epai , whe e egene a ion and ull unc ionali y can
ake up o 4 mon hs [
18
]. Addi ionally, PCL can be combined wi h o he ma e ials o
bioac i e agen s o u he enhance i s biological p ope ies and acili a e issue in eg a ion.
Calejo e al. in es iga ed PCL-based sca olds and ound ha adding hyd oxyapa i e
(HAp) pa icles enhanced cell p oli e a ion and p omo ed an os eogenic pheno ype in
Appl. Sci. 2025,15, 2301 3 o 19
human adipose-de i ed s em cells [
19
]. The s udy also showed ha a ying ilamen
diame e by adjus ing ex ac ion speed signi ican ly imp o ed he mechanical p ope ies,
wi h highe speeds yielding be e pe o mance. Simila ly, Gu lek e al. assessed he
syn hesis o PCL ibe s p oduced by elec ospinning, highligh ing he ma e ial’s capaci y
o cell adhesion and p oli e a ion, al hough he mechanical p ope ies o he elec ospun
PCL ilamen s we e somewha weake compa ed o na i e ACL issue [
20
]. These s udies
unde sco e he e sa ili y o PCL and i s abili y o be cus omized o mee he speci ic
mechanical and biological equi emen s o ligamen epai .
In addi ion o PCL, cellulose nanoc ys als (CNC) ha e a ac ed conside able in e es
as ein o cing agen s in polyme ic sys ems due o hei high su ace a ea, excellen mechani-
cal p ope ies, and biodeg adabili y [
21
,
22
]. CNC, de i ed om enewable esou ces, o e s
a sus ainable op ion o enhancing he mechanical s eng h o polyme sca olds wi hou
comp omising hei biocompa ibili y [
6
,
23
–
25
]. CNC has been shown o imp o e he ensile
s eng h, elas ici y, and s uc u al s abili y o polyme ic sca olds, making hem ideal o
load-bea ing applica ions such as ligamen epai . Liu e al. conduc ed a no able s udy
whe e CNC was inco po a ed in o sodium algina e ibe s using a we -spinning p ocess,
esul ing in signi ican imp o emen s in ensile s eng h and elonga ion a he b eak o he
ilamen s [26]. The in eg a ion o CNC in o PCL sca olds ep esen s a p omising s a egy
o de elop s uc u es wi h enhanced mechanical p ope ies while main aining biocompa i-
bili y [
27
,
28
]. CNC has been widely used as a subs ance conduci e o ob aining ilamen s
wi h dimensions on he mic ome e scale and imp o ed mechanical
p ope ies [26,29–31]
.
Rinoldi e al. emphasized ha he we -spinning echnique, despi e i s limi a ions in p oduc-
ing ilamen s p ima ily a he mic oscale, o e s se e al ad an ages, including he abili y
o con ol ilamen o ien a ion and sca old a chi ec u e, which a e c i ical o mimicking
he mechanical beha io o ligamen s and endons [
32
]. Such p ecise con ol is i al o
eplica ing he hie a chical o ganiza ion o na i e issues, whe e ilamen alignmen plays a
key ole in load dis ibu ion and ensile s eng h.
Fu he mo e, ex ile enginee ing echniques p o ide he ools necessa y o p oduce
ilamen -based s uc u es ha mimic he ex acellula ma ix (ECM) and p omo e cell
g ow h and issue egene a ion [
33
–
35
]. B aided and wo en s uc u es, o ins ance, ha e
been success ully used o de elop ACL sca olds wi h mechanical p ope ies compa able
o na i e issue [
36
]. These echniques allow o he p oduc ion o sca olds wi h unable
mechanical p ope ies, con olled po osi y, and op imal ilamen o ien a ion, acili a ing cell
p oli e a ion and mig a ion. The sca olds p oduced by hese echniques show enhanced
mechanical p ope ies by imp o ing load dis ibu ion and esis ance o de o ma ion. In
he speci ic case o b aided s uc u es, hey p o ide inc eased ensile s eng h and ac u e
esis ance as he in e wined ibe s dis ibu e s ess e enly and p e en c ack p opaga-
ion [
37
]. The abili y o p ecisely con ol he design o he ma e ial, including ilamen
o ien a ion and po e size, is c ucial in c ea ing sca olds ha suppo cell p oli e a ion
and main ain s uc u al in eg i y du ing issue egene a ion [
38
]. Combining ad anced
ab ica ion me hods wi h ma e ial inno a ions, such as PCL and CNC, holds immense
po en ial o add essing he challenges associa ed wi h ACL epai and ad ancing he ield
o issue enginee ing.
In his s udy, we de eloped and e alua ed PCL-based ib ous sca olds ein o ced
wi h CNC using he we -spinning echnique. The inco po a ion o CNC was designed o
enhance he mechanical and biological p ope ies o PCL sca olds, add essing he chal-
lenges associa ed wi h eplica ing he s eng h and s uc u e o na i e ACL issue. An
au oma ed collec o was de eloped and op imized o ensu e ilamen s e ching and diam-
e e educ ion. This me hodology, which combines a non-des uc i e ab ica ion p ocess,
an au oma ed collec o enabling he p ope ilamen , and he selec ion o biocompa ible
Appl. Sci. 2025,15, 2301 4 o 19
polyme s, esul s in a ully biocompa ible sys em wi h enhanced mechanical p ope ies.
The op imized PCL/CNC ilamen s we e cha ac e ized by SEM, TGA, and mechanical
es s and used on he de elopmen o 3D b aided s uc u es ha mimic ACL s uc u e.
Cy o oxici y o he p oduced ilamen s and p oli e a ion es s on he inal s uc u es we e
also pe o med.
2. Ma e ials and Me hods
2.1. Ma e ials
Polycap olac one (PCL) pelle s (molecula weigh o 80,000 g/mol) we e pu chased
om Sigma–Ald ich (S . Louis, MO, USA). Cellulose nanoc ys als (CNC), used as ein-
o cing agen s, we e sou ced om CelluFo ce (Mon eal, QC, Canada). Me hanol (MeOH),
chlo o o m (CHF), ace one, and N,N-Dime hyl o mamide (DMF) we e ob ained om
Sigma–Ald ich and used as sol en s in he p epa a ion o polyme solu ions o in he
coagula ion ba h. All eagen s and sol en s we e used as ecei ed.
2.2. P epa a ion o he PCL and PCL/CNC Polyme Solu ion
Fo he p epa a ion o PCL solu ions, di e en PCL concen a ions (5%, 10%, 15%,
20%, 25%, 30%, and 35% w/ ) and sol en s (CHF, DMF, and ace one) we e e alua ed. Fo
he dissolu ion o PCL, he polyme was g adually added o each sol en unde cons an
s i ing a 30
◦
C un il a homogeneous solu ion was achie ed. To ein o ce he PCL ma ix,
CNC was inco po a ed in o he op imized PCL solu ion a concen a ions o 1%, 2%, and
3% (w/ ). CNC was added g adually a oom empe a u e o p e en agglome a ion,
ollowed by igo ous s i ing o 24 h o ensu e e en dispe sion o he CNC pa icles wi hin
he polyme ma ix. The inal PCL/CNC solu ions we e s o ed un il u he use in he
we -spinning p ocess.
2.3. We -Spinning P ocess
The we -spinning p ocess was employed o p oduce ib ous PCL and PCL/CNC
sca olds using a pump om New E a Pump Sys ems Inc. Solu ions we e ex uded h ough
di e en needles o a ying diame e s (0.41 mm, 0.61 mm, and 0.84 mm) in o a coagula ion
ba h (MeOH). O he pa ame e s, such as he low a e (1 o 9 mL/h), d ying empe a u e
( oom empe a u e o 40
◦
C), and du a ion (10 min o 2 h), we e es ed and op imized. An
au oma ed collec o was de eloped and op imized o ensu e uni o m ilamen s e ching
and imp o ed da a eliabili y. The op imiza ion p ocess in ol ed a obus s uc u e wi h
adjus able olle s and an elec ical sys em, including a mo o and speed egula o , o
main ain cons an ension. The new au oma ed collec o , equipped wi h adjus able olle s
and a mo o ized ension con ol sys em, acili a ed he s e ching o he ilamen s du ing
collec ion, enabling he de elopmen o hinne and mo e uni o m ilamen s (Figu e 1a,b).
2.4. B aiding P ocess
To mimic he ib ous a chi ec u e o he ACL, b aided s uc u es we e ab ica ed using
a T enz–Expo model 16/100 e ical b aiding machine (T enz-Expo S.A., San pedo ,
Spain), which can accommoda e up o 16 bobbins (Figu e 1c). We -spun PCL/CNC ila-
men s we e wound on o bobbins using a T enz–Expo model PR/810 pa allel winding
machine be o e he b aiding p ocess. An app oach using wis ed bundles o 10 ilamen s
pe bobbin was adop ed. The b aided s uc u es we e p oduced by in e lacing ya ns om
16 bobbins in bo h leng hwise and c osswise di ec ions, o ming ubula s uc u es. The
machine’s speed was adjus ed o con ol he b aiding angle and he s uc u al p ope ies
o he esul ing sca olds. Fou di e en ubula s uc u es we e p oduced, a ying in
co e diame e s: 0 mm (compac ), 2 mm, 4 mm, and 6 mm. The co e was empo a ily
Appl. Sci. 2025,15, 2301 5 o 19
inse ed du ing b aiding o main ain he desi ed diame e and was subsequen ly emo ed.
The s uc u es we e hea - ea ed a 40
◦
C o 30 min o s abilize hei shape a e he co e
emo al, ensu ing ha he b aided con igu a ion was p ese ed.
Appl. Sci. 2025, 15, x FOR PEER REVIEW 5 o 20
Figu e 1. (a) We -spinning appa a us; (b) PCL/CNC we -spun ilamen ; and (c) T enz–Expo model
16/100 e ical b aiding machine and PR/810 pa allel winding machine.
2.4. B aiding P ocess
To mimic he ib ous a chi ec u e o he ACL, b aided s uc u es we e ab ica ed us-
ing a T enz–Expo model 16/100 e ical b aiding machine (T enz-Expo S.A.,
San pedo , Spain), which can accommoda e up o 16 bobbins (Figu e 1c). We -spun
PCL/CNC ilamen s we e wound on o bobbins using a T enz–Expo model PR/810 pa -
allel winding machine be o e he b aiding p ocess. An app oach using wis ed bundles o
10 ilamen s pe bobbin was adop ed. The b aided s uc u es we e p oduced by in e lac-
ing ya ns om 16 bobbins in bo h leng hwise and c osswise di ec ions, o ming ubula
s uc u es. The machine’s speed was adjus ed o con ol he b aiding angle and he s uc-
u al p ope ies o he esul ing scaffolds. Fou diffe en ubula s uc u es we e p o-
duced, a ying in co e diame e s: 0 mm (compac ), 2 mm, 4 mm, and 6 mm. The co e was
empo a ily inse ed du ing b aiding o main ain he desi ed diame e and was subse-
quen ly emo ed. The s uc u es we e hea - ea ed a 40 °C o 30 min o s abilize hei
shape a e he co e emo al, ensu ing ha he b aided con igu a ion was p ese ed.
2.5. Cha ac e iza ion Me hods
2.5.1. Mic oscopy Analysis
Mic oscopy analysis was pe o med using a Leica DM750 M Op ical Mic oscope
(Leica Mic osys ems, We zla , Ge many) o obse e he su ace mo phology o he we -
spun ilamen s and b aided s uc u es. Mic oscopy images we e used o assess he diam-
e e o he ilamen s by measu ing hei wid h a 100 diffe en poin s. These measu emen s
we e hen analyzed o calcula e he mean diame e and s anda d de ia ion, assuming a
Gaussian dis ibu ion model o s a is ical consis ency. Fo mo e de ailed su ace analysis,
Field Emission Scanning Elec on Mic oscopy (FESEM) was conduc ed using a Phenom
P oX desk op mic oscope (The mo Fishe Scien i ic, Wal ham, MA, USA). Samples we e
moun ed on aluminum s ubs wi h conduc i e ca bon ape and analyzed wi hou coa ing
Figu e 1. (a) We -spinning appa a us; (b) PCL/CNC we -spun ilamen ; and (c) T enz–Expo model
16/100 e ical b aiding machine and PR/810 pa allel winding machine.
2.5. Cha ac e iza ion Me hods
2.5.1. Mic oscopy Analysis
Mic oscopy analysis was pe o med using a Leica DM750 M Op ical Mic oscope
(Leica Mic osys ems, We zla , Ge many) o obse e he su ace mo phology o he we -
spun ilamen s and b aided s uc u es. Mic oscopy images we e used o assess he diame e
o he ilamen s by measu ing hei wid h a 100 di e en poin s. These measu emen s
we e hen analyzed o calcula e he mean diame e and s anda d de ia ion, assuming a
Gaussian dis ibu ion model o s a is ical consis ency. Fo mo e de ailed su ace analysis,
Field Emission Scanning Elec on Mic oscopy (FESEM) was conduc ed using a Phenom
P oX desk op mic oscope (The mo Fishe Scien i ic, Wal ham, MA, USA). Samples we e
moun ed on aluminum s ubs wi h conduc i e ca bon ape and analyzed wi hou coa ing
unde a ol age o 5 kV. Images we e cap u ed a di e en magni ica ions o assess ilamen
uni o mi y, c oss-sec ional s uc u e, and su ace cha ac e is ics o he PCL and PCL/CNC
ilamen s, as well as b aided s uc u es.
2.5.2. The mog a ime ic Analysis (TGA)
The mal s abili y and deg ada ion p o iles o he PCL and PCL/CNC ilamen s we e
analyzed using a STA 7200 SCANSCI he mog a ime ic analyze (Hi achi High-Tech,
Tokyo, Japan). Samples we e hea ed om 30
◦
C o 600
◦
C a a a e o 10
◦
C/min unde a
ni ogen a mosphe e. The weigh loss and de i a i e weigh loss cu es (DTG) we e used
o de e mine he deg ada ion s ages and he mal beha io o he ilamen s.
Appl. Sci. 2025,15, 2301 6 o 19
2.5.3. Mechanical P ope ies Tes ing
Tensile es s we e conduc ed o e alua e he mechanical p ope ies o he we -spun
PCL and PCL/CNC ilamen s. Tes ing was pe o med using a Houns ield Tinius Olsen
H100KS machine (Tinius Olsen, Redhill, UK) equipped wi h a 250 N load cell. Filamen
samples o 70 mm in leng h we e es ed a a s ain a e o 25 mm/min ollowing ASTM
D5035 [
39
] guidelines. Each sample was es ed in iplica e o ensu e ep oducibili y. Key
mechanical pa ame e s, including ensile s eng h, s ain a b eak, and Young’s modulus,
we e de i ed om s ess–s ain cu es. The ensile s eng h co esponds o he maximum
s ess eco ded be o e he ilamen ac u ed. The elonga ion a b eak was de e mined om
he o al s ain a ailu e. The s ess–s ain cu es we e ob ained by eco ding he applied
o ce and he co esponding elonga ion o he ilamen un il ailu e. S ess (
σ
), s ain (
ε
),
and Young’s modulus (E) we e calcula ed using Equa ions (1)–(3):
σ=
F
A(1)
ε=
∆L
L0(2)
E=
∆σ
∆ε(3)
whe e Fis he applied o ce (N), Ais he ini ial c oss-sec ional a ea o he ilamen (mm
2
),
∆
Lis he change in ilamen leng h (mm), L
0
is he ini ial leng h o he sample (mm), and
∆σand ∆εa e he changes in s ess and s ain wi hin he linea elas ic de o ma ion ange.
2.6. Cy o oxici y and P oli e a ion E alua ion
2.6.1. Samples P epa a ion
P io o he assays, he ilamen s and s uc u es unde wen s e iliza ion h ough a
se ies o washing s eps [
40
]. They we e i s washed wice wi h e hanol, ollowed by wo
washes wi h wa e , wo washes wi h PBS (1
×
) con aining 1% ( / ) penicillin/s ep omycin,
and wo addi ional washes wi h wa e . Each washing s ep in ol ed 20 min o incuba ion.
A e d ying, he ilamen s we e s e ilized wi h ul a iole (UV) adia ion o 60 min.
2.6.2. Cell Cul u e
BJ-5 a cell line (no mal human ib oblas s immo alized by o e exp ession o elom-
e ase) was cul u ed in ou pa s o DMEM (powde ), con aining L-glu amine, D-glucose,
sodium bica bona e, and one pa medium 199, supplemen ed wi h 5% FBS, 1% ( / )
penicillin/s ep omycin solu ion, and 10 µg/mL o hyg omycin.
Cellula subcul u es we e pe o med when con luence eached alues close o 80–90%.
BJ-5 a cells we e main ained in 25 cm
2
issue cul u e lasks in an incuba o a 37
◦
C in a
humidi ied a mosphe e wi h 5% CO
2
. The cell cul u e medium was enewed wo imes pe
week. Fo subcul u es and pla ing, he adhe en cells we e de ached wi h ypsin solu ion
0.05%, and esh medium was added in o de o neu alize he ypsin. The cell suspension
was cen i uged o 5 min a 160
×
g. The supe na an was disca ded, and a esh medium
was added o ob ain a new cell suspension. The cell suspension was loaded in a Neubaue
chambe , and he concen a ion o cells was es ima ed [41].
2.6.3. Cy o oxici y E alua ion
The cy o oxici y o he ilamen s o PCL wi h and wi hou CNC was assessed by
indi ec con ac by exposing BJ-5 a cells o condi ioned media con aining ilamen ilm
deg ada ion p oduc s and leachables.
Appl. Sci. 2025,15, 2301 7 o 19
The ilamen s we e s e ilized by UV o 1 h p io o incuba ion wi h he cul u e media.
The condi ioned media we e ob ained by incuba ing he s e ilized ilamen s in 2 mL o
medium a 37
◦
C in a humidi ied a mosphe e o 5% CO
2
o 24 h. Cells we e seeded a a
densi y o 10
×
10
3
cells/100
µ
L/well on a 96-well issue cul u e pla e he day be o e he
expe imen s. The cells we e hen exposed o he condi ioned medium in di e en pe cen ages
(100%, 50%, and 25%) and we e u he incuba ed a 37
◦
C in a humidi ied a mosphe e o 5%
CO
2
. Cells incuba ed wi h dime hyl sulphoxide (DMSO, 30%) we e used as con ol o dea h,
and cells g own wi h cul u e medium we e used as con ol o li e. A he end o 24 and 48 h
o con ac , he me abolic iabili y was assessed by MTS iabili y assay (P omega, Alexand ia,
Aus alia) ollowing he manu ac u e ’s p o ocol [
42
]. The educ ion in MTS by iable cells
was measu ed wi h a 96-well pla e eade a 570 nm in a mic opla e eade (Syne gy H11,
BIOTEK, Winooski, VT, USA). Rela i e iabili y was calcula ed in ela ion o sol en con ol
and exp essed g aphically. Each sample was assayed in iplica e.
2.6.4. Cell P oli e a ion Assays
The abili y o ilamen s composed o PCL and CNC o suppo cell p oli e a ion was
assessed using BJ5- a ib oblas s and he esazu in-based Xpe Blue Cell Viabili y Assay
(GRISP, Po ugal). A cell suspension was p epa ed a a concen a ion o 1
×
10
6
cells/mL,
and 5
×
10
4
cells we e seeded on o ilamen s ha had been p econdi ioned in cul u e media
wi hou FBS o 24 h. The condi ioned ilamen s we e placed in a 12-well cul u e pla e,
and hal o he cells (25
µ
L o cell suspension) we e added o he ilamen s, ollowed by
a 1 h incuba ion. A e wa d, an addi ional 25
µ
L o cell suspension was applied, and he
ilamen s we e incuba ed o 2 mo e hou s [43].
Once seeded, he ilamen s we e gen ly ans e ed o new wells, and esh comple e
cul u e media we e added. Hal o he cul u e media was e eshed e e y 48 h. A 24 and
72 h pos -seeding, he ilamen s we e mo ed o new wells, and esh media con aining
esazu in we e added, in acco dance wi h he manu ac u e ’s ins uc ions. The educ ion
o esazu in by iable cells was measu ed using a mic opla e eade (BioTek Syne gy
H11, Agilen , San a Cla a, CA, USA) a an exci a ion/emission (Ex/Em) wa eleng h o
560/590 nm. Cell p oli e a ion was de e mined using Equa ion (4).
In ensi y Tx
In ensi y T0
×100 −100, (4)
whe e T
x
ep esen s he ime poin s a 48, 72, and 144 h, and T
0
co esponds o he 24 h
ime poin . All samples we e p epa ed in iplica e.
3. Resul s and Discussion
3.1. Op imiza ion o PCL and PCL/CNC Filamen s P oduced by We -Spinning
The we -spinning p ocess was op imized by a ying key pa ame e s, including poly-
me concen a ion, needle diame e , low a e, and he composi ion o he coagula ion ba h,
o achie e uni o m PCL and PCL/CNC ilamen s wi h desi able mechanical p ope ies.
Ini ial es s we e conduc ed wi h PCL concen a ions anging om 5% o 35% (w/ ) in di -
e en sol en s, including CHF, DMF, wa e , and ace one. Despi e es ing di e en sol en s,
CHF was selec ed as he op imal sol en due o i s supe io abili y o dissol e PCL and
o m homogeneous solu ions. I was obse ed ha PCL concen a ions below 25% we e
insu icien o p oduce con inuous ilamen s, while 35% PCL ilamen s we e mo e i egula ,
wi h he e ogeneous diame e s and dis ibu ions. The e o e, concen a ions o 25% and 30%
PCL yielded mo e consis en ilamen s and we e used in he condi ion’s op imiza ion.
Flow a es anging om 1 o 9 mL/h we e es ed wi h needles o a ying diame e s
(0.41, 0.61, and 0.84 mm). The op imal needle diame e o 25% PCL was ound o be
Appl. Sci. 2025,15, 2301 8 o 19
0.41 mm
, while he op imal low a e was 4 mL/h. Rega ding 30% PCL, a needle diame e
o 0.61 mm and a low a e o 6 mL/h esul ed in he mos uni o m ilamen mo phology.
These condi ions minimized he o ma ion o beads and inconsis encies along he ilamen
leng h, leading o imp o ed ilamen homogenei y. The d ying p ocess was also e alua ed
and op imized. I was ound ha he op imal d ying condi ions we e 30 min a 40 ◦C.
All solu ions we e ex uded in o a MeOH coagula ion ba h and collec ed on an au o-
ma ed collec o . MeOH was selec ed as he coagula ion ba h a e es ing mul iple sol en s
(wa e , e hanol), as i p o ided he mos uni o m ibe o ma ion. The as sol en exchange
in MeOH allowed o quick polyme solidi ica ion, p e en ing ibe de ec s and agg e-
ga ion, which was no achie ed wi h o he sol en s. The collec o was de eloped and
op imized o ensu e uni o m ilamen s e ching and was based on a s uc u e consis ing
o a collec ing olle posi ioned below and wo adjus able olle s ha could be placed
a di e en heigh s and dis ances be o e he collec ion poin (Figu e 1a). Mo eo e , he
collec ing olle included a mo o and speed egula o o main ain cons an ension.
The op imiza ion p ocess in ol ed sys ema ically es ing di e en posi ions o he
wo adjus able olle s and e alua ing hei e ec on ilamen s e ching. I was obse ed
ha he bes con igu a ion was achie ed by using he wo guiding olle s placed in he
posi ions shown on Figu e 1a. The de eloped collec o allowed he s e ching o PCL
ilamen s, eaching ibe diame e s as low as 30
µ
m. Fo 25% PCL, a ilamen diame e o
229.44
±
7.30
µ
m was ob ained wi hou s e ching. Wi h he use o he au oma ed collec o ,
his alue dec eased o 42.03
±
3.19
µ
m, p o ing he impo ance o ilamen s e ching.
Rega ding 30% PCL, diame e s o 255.42
±
7.38
µ
m and 29.79
±
1.24
µ
m we e ob ained o
he ilamen s wi hou and wi h s e ching, espec i ely.
The inco po a ion o CNC was pe o med on he 25 and 30% (w/ ) op imized PCL
solu ion. A de ailed analysis o he op imiza ion o he mix u e be ween he componen s
unde s udy was conduc ed, as well as he e ec o a ying he pe cen age o CNC (1, 2,
and 3% (w/ )). The op imized PCL solu ions (25 and 30% (w/ )) we e ein o ced wi h
he di e en pe cen ages o CNC, which we e added g adually o p e en agglome a ion.
The solu ions we e s i ed o 24 h and hen we spun ollowing he same condi ions. All
ilamen s we e analyzed by mic oscopic echniques, and he mo phology and dimensions
o each ilamen we e s udied. While pu e PCL ilamen s exhibi ed a smoo h su ace, he
PCL/CNC ilamen s demons a ed a mo e ex u ed mo phology due o he p esence o
CNC. This su ace oughness is a ibu ed o he pa ial exposu e o CNC on he ilamen
su ace, which can po en ially enhance cell a achmen in biological applica ions. The
solu ion pa ame e s, we -spinning condi ions, and he calcula ed ilamen diame e s a e
de ailed in Table 1.
Table 1. Solu ion pa ame e s (PCL and CNC pe cen age), we -spinning condi ions (needle diame e
and low a e), and ilamen a e age diame e s and s anda d de ia ions o he ilamen s ob ained.
Sample Polyme Pe cen age
(%)
We -Spinning Condi ions Mean Diame e
(µm)
Needle
Diame e (mm)
Flow Ra e
(mL/h)
F1 25% PCL/1% CNC
0.41 4
50.53 ±1.04
F2 25% PCL/2% CNC 52.67 ±1.09
F3 25% PCL/% CNC 55.00 ±1.32
F4 30% PCL/1% CNC
0.61 6
49.88 ±1.09
F5 30% PCL/2% CNC 50.26 ±1.35
F6 30% PCL/3% CNC 53.57 ±1.64
Appl. Sci. 2025,15, 2301 9 o 19
A mechanical analysis o he six ilamen s was conduc ed by pe o ming s ess–s ain
cu es. Fo each ilamen , 10 eplica es we e es ed. All he s ess–s ain cu es showed
a con igu a ion simila o he cu e ep esen ing a no mal endon when subjec ed o a
mechanical ensile es . The alues o maximum ension, maximum s ess de o ma ion,
b eaking poin , s ain, and Young’s modulus we e assessed and a e de ailed in Table 2.
Table 2. Maximum ension, maximum s ess de o ma ion, b eaking poin , s ain, and Young’s
modulus o he ilamen s ob ained.
Sample
Tensile
S eng h
(MPa)
Maximum S ess
De o ma ion (%)
B eaking
Poin (MPa) S ain (%)
Young’s
Modulus
(GPa)
R2
F1 70.28 1130.36 69.86 1129.72 0.18 0.95
F2 131.86 773.28 129.12 786.00 0.19 0.92
F3 201.34 750.30 200.87 762.47 0.21 0.92
F4 80.01 957.29 79.26 858.04 0.17 0.96
F5 169.10 947.16 161.40 956.22 0.22 0.92
F6 277.65 904.40 272.68 913.21 0.27 0.95
A e a comp ehensi e analysis o he esul s ob ained, he maximum s ess de o -
ma ion alues s and ou as hey p o ed o be ema kably high, highligh ing one o he
mos dis inc i e cha ac e is ics o he PCL polyme , i.e., i s elonga ion capaci y. Howe e ,
i was obse ed ha he inco po a ion o CNC in he o mula ions led o a educ ion in
de o ma ion as a esul o he g ea e igidi y p o ided o he ilamen s by he p esence
o CNC. E en so, he ilamen s main ained subs an ial de o ma ion alues. Acco ding
o he li e a u e, heal hy endons and ligamen s ope a e unde ailu e loads be ween 7%
and 40% [
32
]. I was ound ha all he op imized o mula ions in his s udy signi ican ly
exceeded hese alues.
In gene al, i can be in e ed ha he addi ion o CNC o PCL solu ions esul s in an
inc ease in ensile s eng h and b eaking poin , leading o he exclusion o o mula ions
wi h lowe pe cen ages o CNC in all op imized solu ions. In addi ion, he alues ob ained
indica e ha inc easing he pe cen age o PCL ansla es in o an inc ease in he ensile
s eng h and b eaking poin , poin ing o a close app oxima ion o he main objec i e when
combined wi h a highe pe cen age o CNC. The esul s ob ained co obo a e he idea ha
CNC plays a signi ican ole in he mechanical ein o cemen o ilamen s.
Young’s modulus alues we e simila o all he op imized ilamen s (0.18–0.27 GPa).
The alues o Young’s modulus ound in he li e a u e o he an e io c ucia e ligamen
(ACL), he pa ella ligamen (PL), and he endon o he iple semi endinosus muscle
(STT) we e ound o be 0.195, 0.426, and 0.163 GPa, espec i ely [
44
]. This co obo a es
he po en ial o he p oduced ilamen s o eplace ACL, wi h simila alues o Young’s
modulus. Pa icula ly, he ilamen p oduced wi h 30% (w/ ) PCL and 3% (w/ ) CNC
shows a Young’s modulus o 0.27 GPa, which ep esen s a 38% inc ease when compa ed o
he ACL alue.
O e all, he op imized condi ions o p oducing uni o m and mechanically obus
PCL/CNC ilamen s in ol ed a 30% (w/ ) PCL concen a ion wi h 3% (w/ ) CNC, a low
a e o 6 mL/h, and a 0.61 mm needle diame e . These condi ions p o ided a balance
be ween ilamen uni o mi y and he desi ed enhancemen in mechanical p ope ies o
ACL issue enginee ing applica ions.
3.2. Cha ac e iza ion o PCL/CNC We -Spun Filamen s
The he mog a ime ic analysis (TGA) was conduc ed o assess he he mal s abili y
and deg ada ion beha iou o he PCL/CNC composi e ilamen s compa ed o pu e PCL
Appl. Sci. 2025,15, 2301 16 o 19
diame e s as low as 30
µ
m. The op imized ilamen s we e p oduced wi h a 30% (w/ ) PCL
concen a ion and 3% (w/ ) CNC, a low a e o 6 mL/h, and a 0.61 mm needle diame e .
The p oduced ilamen s showed an a e age diame e o 53.57
µ
m and demons a ed
good mechanical p ope ies, achie ing a balance be ween ensile s eng h and lexibili y
(Young’s modulus o 0.27 GPa), making i a sui able candida e o issue enginee ing
applica ions. The cy o oxici y esul s indica ed ha he 30% PCL/3% CNC o mula ion did
no elici any ad e se e ec s on ib oblas iabili y, suppo ing i s po en ial o sa e use in
biomedical applica ions.
The s udy also explo ed he de elopmen o b aided 3D s uc u es om he op imized
ilamen s, aiming o eplica e he ib ous o ganiza ion o na u al ligamen s. The use o
wis ed ilamen bundles and a ying co e sizes allowed o he c ea ion o s uc u es wi h
di e en mechanical and dimensional p ope ies wi hou changing he co e in eg i y o
he indi idual ilamen s. P oli e a ion es s demons a ed an 81% success a e, indica ing a
p omising le el o cellula compa ibili y and sugges ing ha he ab ica ed s uc u es ha e
signi ican po en ial o applica ions in issue enginee ing.
The 30% PCL/3% CNC ilamen s and hei de i ed b aided s uc u es show p omise
as sca olds o ACL epai . The combina ion o PCL, a biodeg adable and biocompa ible
polyme , wi h CNC, a ein o cing agen wi h simila p ope ies, enables he de elopmen
o a ully biodeg adable sys em wi h enhanced mechanical pe o mance. By employing
an op imized we -spinning p ocess and a b aiding echnique, his app oach in eg a es
ul a- hin ilamen s in o 3D s uc u es wi h supe io ensile s eng h and Young’s modulus,
closely ma ching he mechanical p ope ies o na i e ACL issue. Fu u e wo k will ocus
on e ining he p oduc ion p ocess o 3D s uc u es, pa icula ly imp o ing he uni o mi y
and mechanical pe o mance o la ge -diame e sca olds and conduc ing
in i o
s udies
o u he alida e hei e icacy o ACL issue enginee ing.
Au ho Con ibu ions: Concep ualiza ion, D.P.F.; me hodology, J.M.R., R.P.C.L.S., D.S. and S.D.T.; o -
mal analysis, R.P.C.L.S., S.D.T. and D.P.F.; in es iga ion, J.M.R., R.P.C.L.S., D.S., S.D.T. and A.R.;
w i ing—o iginal d a p epa a ion, J.M.R., R.P.C.L.S. and S.D.T.; w i ing— e iew and edi ing,
R.P.C.L.S., S.D.T. and D.P.F.; supe ision, D.P.F.; p ojec adminis a ion, D.P.F. and R.F.; unding acqui-
si ion, D.P.F. and R.F. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding: This esea ch was unded by he Eu opean Regional De elopmen Fund h ough he
Ope a ional Compe i i eness P og am and he Na ional Founda ion o Science and Technology
o Po ugal (FCT) unde he p ojec s UID/CTM/00264/2020 o he Cen e o Tex ile Science
and Technology (2C2T) on i s componen s base (h ps://doi.o g/10.54499/UIDB/00264/2020, ac-
cessed on 10 Janua y 2025) and p og am (h ps://doi.o g/10.54499/UIDP/00264/2020, accessed
on 10 Janua y 2025), unde he scope o he Cen e o Biological Enginee ing (CEB) s a egic
unding wi h e e ence UIDB/04469/2020 (h ps://doi.o g/10.54499/UIDB/04469/2020, accessed
on 10 Janua y 2025), and by LABBELS—Associa e Labo a o y in Bio echnology, Bioenginee ing
and Mic oelec omechanical Sys ems, LA/P/0029/2020. The au ho s acknowledge he inancial
suppo om he in eg a ed p ojec GIATEX—Ges ão In eligen e da Água na ITV (In es men
RE-C05-i01.01—Mobilizing Agendas/Alliances o Business Inno a ion), p omo ed by he Reco -
e y and Resilience Plan (RRP), Nex Gene a ion EU, o he pe iod om 2022–2025. Diana P.
Fe ei a is hank ul o CEECIND/02803/2017, unded by Na ional Funds h ough FCT/MCTES
(h ps://doi.o g/10.54499/CEECIND/02803/2017/CP1458/CT0003, accessed on 10 Janua y 2025).
A.R. hanks FCT o i s con ac unde he CEEC-Indi idual—4 h Edi ion wi h he e e ence
2021.02803.CEECIND.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Appl. Sci. 2025,15, 2301 17 o 19
Da a A ailabili y S a emen : The o iginal con ibu ions p esen ed in his s udy a e included in he
a icle. Fu he inqui ies can be di ec ed o he co esponding au ho s.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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