Progress in 3D Bioprinting Technology for Osteochondral Regeneration

Ci a ion: La uen e-Me chan, M.;
Ruiz-Alonso, S.; Ga cía-Villén, F.;
Gallego, I.; Gál ez-Ma ín, P.;
Saenz-del-Bu go, L.; Ped az, J.L.
P og ess in 3D Biop in ing
Technology o Os eochond al
Regene a ion. Pha maceu ics 2022,14,
1578. h ps://doi.o g/10.3390/
pha maceu ics14081578
Academic Edi o : Ca men Fe e o
Recei ed: 28 June 2022
Accep ed: 28 July 2022
Published: 29 July 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 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/).
pha maceu ics
Re iew
P og ess in 3D Biop in ing Technology o
Os eochond al Regene a ion
Ma kel La uen e-Me chan 1,2,3 , Sand a Ruiz-Alonso 1,2,3 , Fá ima Ga cía-Villén1,2,3 , Idoia Gallego 1,2,3 ,
Pa icia Gál ez-Ma ín4, Lau a Saenz-del-Bu go 1,2,3,* and Jose Luis Ped az 1,2,3,*
1NanoBioCel G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque
Coun y (UPV/EHU), Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain;
[email p o ec ed] (M.L.-M.); [email p o ec ed] (S.R.-A.); a ima.ga [email p o ec ed] (F.G.-V.);
[email p o ec ed] (I.G.)
2Biomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN),
Heal h Ins i u e Ca los III, Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain
3Bioa aba, NanoBioCel Resea ch G oup, 01009 Vi o ia-Gas eiz, Spain
4R&D Animal and Human Heal h, Bioibé ica S.A.U., 08029 Ba celona, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed] (L.S.-d.-B.); [email p o ec ed] (J.L.P.)
Abs ac :
Os eochond al inju ies can lead o os eoa h i is (OA). OA is cha ac e ized by he p o-
g essi e deg ada ion o he ca ilage issue oge he wi h bone issue u no e . Consequen ly, join
pain, in lamma ion, and s i ness a e common, wi h join immobili y and dys unc ion being he mos
se e e symp oms. The inc ease in he age o he popula ion, along wi h he inc ease in isk ac o s
such as obesi y, has led OA o he o e on o disabling diseases. In addi ion, i no only has an
inc easing p e alence, bu is also an economic bu den o heal h sys ems. Cu en ea men s a e
ocused on elie ing pain and in lamma ion, bu hey become ine ec i e as he disease p og esses.
The e o e, new he apeu ic app oaches, such as issue enginee ing and 3D biop in ing, ha e eme ged.
In his e iew, he ad an ages o using 3D biop in ing echniques o os eochond al egene a ion a e
desc ibed. Fu he mo e, he bioma e ials, cell ypes, and ac i e molecules ha a e commonly used
o hese pu poses a e indica ed. Finally, he mos ecen p omising esul s o he egene a ion o
ca ilage, bone, and/o he os eochond al uni h ough 3D biop in ing echnologies a e conside ed,
as his could be a easible he apeu ic app oach o he ea men o OA.
Keywo ds:
3D biop in ing; os eoa h i is; issue enginee ing; egene a i e medicine; ca ilage; bone
1. In oduc ion
The aging o he popula ion, oge he wi h he inc ease in he p e alence o isk ac o s
such as obesi y, physical inac i i y, and ex eme exe cise, has placed os eoa icula diseases
in he ocus o medicine. Os eochond al de ec s a e cha ac e ized by ca ilage dis up ion
oge he wi h bone damage. Join aumas and inju ies a e he mos common causes o
os eochond al de ec s [
1
]. Ne e heless, join umo s and in ec ions can also be he igge s
o os eochond al damage [
2
,
3
]. Fu he mo e, he a e disease os eochond i is dissecans
should be also aken in o conside a ion [
4
]. Howe e , among hem, os eoa h i is (OA) has
gained no o ie y by becoming he hi d mos common condi ion associa ed wi h disabili y,
a e demen ia and diabe es [5]. In ac , i is es ima ed ha 250 million people a e a ec ed
wo ldwide, and ha he p opo ion o he popula ion wi h an OA diagnosis will inc ease
by 3% by he yea 2032 [
6
]. This inc ease in p e alence will no only wo sen he quali y o
li e o he a ec ed popula ion, bu will also en ail an economic cos o heal hca e sys ems.
1.1. P e alence and Economic Bu den
OA is a disease o he join s such as he knee, hip, and hand ha a ec s 7% o he wo ld
popula ion [
7
]. Fo example, in he US alone, 37% o people o e he age o 65 su e om
Pha maceu ics 2022,14, 1578. h ps://doi.o g/10.3390/pha maceu ics14081578 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2022,14, 1578 2 o 33
his disease [
8
]. Age is he mos impo an isk ac o o he onse o he disease. Likewise,
obesi y is conside ed o be ano he impo an ac o ha con ibu es o he appea ance o
OA. In ac , due o he aging o he popula ion and he ise in obesi y a es, he p e alence o
OA has isen by 48% in he las 30 yea s [
9
]. Fu he mo e, i is es ima ed ha he p e alence
o OA in he popula ion aged o e 45 will inc ease in he coming yea s. In addi ion, i has
been shown ha ha d wo k ac i i ies, high-impac spo s, and gene ics can also in luence
he appea ance o OA in younge indi iduals [6].
Gi en he inc ease in he disease’s incidence, he economic cos s associa ed wi h OA
mus be conside ed as ano he p oblem. In he US, i is es ima ed ha he economic bu den
anges be ween USD 3.4 and 13.2 billion pe yea [
8
]. Globally, i is es ima ed ha he
medical cos s associa ed wi h OA in ich coun ies a e be ween 1 and 2.5% o he g oss
domes ic p oduc [
6
,
10
]. Howe e , indi ec cos s due o wo k loss, medical lea e, and
p ema u e e i emen s could inc ease he economic bu den [
11
]. In ac , acco ding o he
Wo ld Heal h O ganiza ion (WHO), OA is in he op 10 diseases ha cause wo k loss due
o disabili y [
12
]. When i comes o indica ing each cos sepa a ely, he e is conside able
disag eemen in he li e a u e due o he lack o uni o m c i e ia ac oss he s udies, he
coun y whe e he s udy was ca ied ou , and he ana omical loca ion and s age o OA [
13
].
Fo ins ance, in Spain, he annual cos is es ima ed a a ound EUR 1500 pe pa ien . In
addi ion, Loza e al. [
13
] conduc ed a b eakdown o he economic expenses associa ed
wi h knee and hip OA in Spain (Figu e 1). Acco ding o hem, di ec cos s we e a ound
86%, which could be sepa a ed in o medical cos s (47%) and nonmedical cos s (39%).
Medical cos s included expenses in e ms o sani a y p o essional ime (22%), hospi al
admissions (13%), medical es s and p obes (7%), and d ug cos s (5%). Nonmedical cos s
we e mainly ela ed o house, wo k, and sel -ca e assis ance (29%), aid se ices (9%),
and pa ien anspo cos s (1%). On he o he hand, indi ec cos s we e es ima ed o
accoun o 14%; 8% wen on assis ance o housewo k, and 6% was due o loss o wo k,
wo kplace absences, and a dec ease in p oduc i i y [
14
]. Ne e heless, he o al cos o
OA d as ically inc eased when he disease was in he se e e s ages and when he pa ien s
equi ed hospi aliza ion [13].
Pha maceu ics 2022, 14, x FOR PEER REVIEW 2 o 34
1.1. P e alence and Economic Bu den
OA is a disease o he join s such as he knee, hip, and hand ha a ec s 7% o he
wo ld popula ion [7]. Fo example, in he US alone, 37% o people o e he age o 65 su e
om his disease [8]. Age is he mos impo an isk ac o o he onse o he disease.
Likewise, obesi y is conside ed o be ano he impo an ac o ha con ibu es o he ap-
pea ance o OA. In ac , due o he aging o he popula ion and he ise in obesi y a es,
he p e alence o OA has isen by 48% in he las 30 yea s [9]. Fu he mo e, i is es ima ed
ha he p e alence o OA in he popula ion aged o e 45 will inc ease in he coming yea s.
In addi ion, i has been shown ha ha d wo k ac i i ies, high-impac spo s, and gene ics
can also in luence he appea ance o OA in younge indi iduals [6].
Gi en he inc ease in he disease’s incidence, he economic cos s associa ed wi h OA
mus be conside ed as ano he p oblem. In he US, i is es ima ed ha he economic bu -
den anges be ween USD 3.4 and 13.2 billion pe yea [8]. Globally, i is es ima ed ha he
medical cos s associa ed wi h OA in ich coun ies a e be ween 1 and 2.5% o he g oss
domes ic p oduc [6,10]. Howe e , indi ec cos s due o wo k loss, medical lea e, and
p ema u e e i emen s could inc ease he economic bu den [11]. In ac , acco ding o he
Wo ld Heal h O ganiza ion (WHO), OA is in he op 10 diseases ha cause wo k loss due
o disabili y [12]. When i comes o indica ing each cos sepa a ely, he e is conside able
disag eemen in he li e a u e due o he lack o uni o m c i e ia ac oss he s udies, he
coun y whe e he s udy was ca ied ou , and he ana omical loca ion and s age o OA
[13]. Fo ins ance, in Spain, he annual cos is es ima ed a a ound EUR 1500 pe pa ien .
In addi ion, Loza e al. [13] conduc ed a b eakdown o he economic expenses associa ed
wi h knee and hip OA in Spain (Figu e 1). Acco ding o hem, di ec cos s we e a ound
86%, which could be sepa a ed in o medical cos s (47%) and nonmedical cos s (39%). Med-
ical cos s included expenses in e ms o sani a y p o essional ime (22%), hospi al admis-
sions (13%), medical es s and p obes (7%), and d ug cos s (5%). Nonmedical cos s we e
mainly ela ed o house, wo k, and sel -ca e assis ance (29%), aid se ices (9%), and pa-
ien anspo cos s (1%). On he o he hand, indi ec cos s we e es ima ed o accoun o
14%; 8% wen on assis ance o housewo k, and 6% was due o loss o wo k, wo kplace
absences, and a dec ease in p oduc i i y [14]. Ne e heless, he o al cos o OA d as ically
inc eased when he disease was in he se e e s ages and when he pa ien s equi ed hos-
pi aliza ion [13].
Due o he inc ease in cases ha no only wo sen he quali y o li e o hose a ec ed
pa ien s, bu also ha e a g ea economic cos , imp o emen o he ea men o his disease
is a necessi y.
Figu e 1. The economic cos o knee and hip OA in Spain. Da a om [14].
Figu e 1. The economic cos o knee and hip OA in Spain. Da a om [14].
Due o he inc ease in cases ha no only wo sen he quali y o li e o hose a ec ed
pa ien s, bu also ha e a g ea economic cos , imp o emen o he ea men o his disease
is a necessi y.
Cu en ea men s a e mainly based on pallia i e d ugs, wi h su ge y being he las
eso , and only in he mos se e e cases. Howe e , as hese ea men s ha e been shown
o be ine ec i e in he majo i y o cases, he e is a necessi y in he scien i ic communi y
o de elop new he apeu ic app oaches. In his ega d, issue enginee ing has d awn
a en ion, since i combines di e en biomedical ields such as cell he apy, nano echnology,
and bioma e ial science [
15
]. Fu he mo e, addi i e manu ac u ing echnologies such as 3D
Pha maceu ics 2022,14, 1578 3 o 33
biop in ing ha e eme ged o acili a e issue enginee ing pu poses in a apid and au oma ic
manne [
16
]. Thus, he bio ab ica ion o unc ional sca olds ha could egene a e damaged
issues is cu en ly a i s peak. Howe e , p io o he ab ica ion o a i icial issues, i is
necessa y o achie e in-dep h knowledge o he os eochond al issue and OA.
1.2. Join Ana omy and Physiology
Inju ies o he os eochond al issue may lead o OA. Thus, OA is a diso de ha
a ec s he whole join [
6
]. Join s a e a eas o a icula ion be ween adjacen bones and
ca ilage o he pu pose o p o iding s abili y and mobili y [
17
]. Figu e 2shows he
schema ic o ganiza ion o he join issue sepa a ed in o wo a eas: ca ilage and bone. A
he same ime, bo h ca ilage and bone ha e di e en laye s wi h hei own composi ion
and cha ac e is ics.
Pha maceu ics 2022, 14, x FOR PEER REVIEW 3 o 34
Cu en ea men s a e mainly based on pallia i e d ugs, wi h su ge y being he las
eso , and only in he mos se e e cases. Howe e , as hese ea men s ha e been shown
o be ine ec i e in he majo i y o cases, he e is a necessi y in he scien i ic communi y o
de elop new he apeu ic app oaches. In his ega d, issue enginee ing has d awn a en-
ion, since i combines di e en biomedical ields such as cell he apy, nano echnology,
and bioma e ial science [15]. Fu he mo e, addi i e manu ac u ing echnologies such as
3D biop in ing ha e eme ged o acili a e issue enginee ing pu poses in a apid and au-
oma ic manne [16]. Thus, he bio ab ica ion o unc ional sca olds ha could egene a e
damaged issues is cu en ly a i s peak. Howe e , p io o he ab ica ion o a i icial is-
sues, i is necessa y o achie e in-dep h knowledge o he os eochond al issue and OA.
1.2. Join Ana omy and Physiology
Inju ies o he os eochond al issue may lead o OA. Thus, OA is a diso de ha a -
ec s he whole join [6]. Join s a e a eas o a icula ion be ween adjacen bones and ca i-
lage o he pu pose o p o iding s abili y and mobili y [17]. Figu e 2 shows he schema ic
o ganiza ion o he join issue sepa a ed in o wo a eas: ca ilage and bone. A he same
ime, bo h ca ilage and bone ha e di e en laye s wi h hei own composi ion and cha -
ac e is ics.
Figu e 2. Schema ic o ganiza ion o os eochond al issue.
Ca ilage is an a ascula , aneu al, and alympha ic issue ha is ound a he end o
long bones [6]. I is composed o highly specialized cells known as chond ocy es and an
ex acellula ma ix (ECM). This ECM is cons i u ed o glycosaminoglycans (GAGs) and
collagens ha allow he e en ion o la ge amoun s o wa e . Conside ing he a ascula
na u e o he ca ilage, his luid no only allows he chond ocy es o be supplied wi h
nu ien s, bu is also esponsible o p o iding esis ance o mechanical comp ession [18].
Consequen ly, ca ilage is a issue wi h signi ican biomechanics. Fo ins ance, he
Young’s modulus o ca ilage is be ween 0.2 and 2 MPa [19]. A he same ime, he a icu-
la ca ilage is mo phologically classi ied in o h ee zones depending on chond ocy e o -
ganiza ion, collagen ib il o ien a ion, and GAG con en .
The supe icial laye is he hinne laye , and p o ec s deepe laye s om shea
s esses. I is cha ac e ized by la e ed chond ocy es, collagen ib ils o ien ed pa allel o
he a icula su ace, and low GAG con en . The middle laye is he hickes laye , and unc-
ions as an ana omical and unc ional zone be ween he supe icial laye and he deep
laye . Chond ocy es a e a low densi y and sphe ical, collagen ib ils a e obliquely o i-
en ed, and he GAG con en is inc eased. This laye is he i s line o esis ance o com-
p essi e o ces. Finally, he e is he deep laye . In his zone, chond ocy e densi y is in-
c eased, and hey a e a anged in columna o ien a ion. Collagen ib ils a e o ien a ed
Figu e 2. Schema ic o ganiza ion o os eochond al issue.
Ca ilage is an a ascula , aneu al, and alympha ic issue ha is ound a he end o
long bones [
6
]. I is composed o highly specialized cells known as chond ocy es and an
ex acellula ma ix (ECM). This ECM is cons i u ed o glycosaminoglycans (GAGs) and
collagens ha allow he e en ion o la ge amoun s o wa e . Conside ing he a ascula
na u e o he ca ilage, his luid no only allows he chond ocy es o be supplied wi h
nu ien s, bu is also esponsible o p o iding esis ance o mechanical comp ession [
18
].
Consequen ly, ca ilage is a issue wi h signi ican biomechanics. Fo ins ance, he Young’s
modulus o ca ilage is be ween 0.2 and 2 MPa [
19
]. A he same ime, he a icula ca ilage
is mo phologically classi ied in o h ee zones depending on chond ocy e o ganiza ion,
collagen ib il o ien a ion, and GAG con en .
The supe icial laye is he hinne laye , and p o ec s deepe laye s om shea s esses.
I is cha ac e ized by la e ed chond ocy es, collagen ib ils o ien ed pa allel o he a ic-
ula su ace, and low GAG con en . The middle laye is he hickes laye , and unc ions
as an ana omical and unc ional zone be ween he supe icial laye and he deep laye .
Chond ocy es a e a low densi y and sphe ical, collagen ib ils a e obliquely o ien ed, and
he GAG con en is inc eased. This laye is he i s line o esis ance o comp essi e o ces.
Finally, he e is he deep laye . In his zone, chond ocy e densi y is inc eased, and hey
a e a anged in columna o ien a ion. Collagen ib ils a e o ien a ed pe pendicula o he
su ace, and he GAG con en is he highes . The deep laye p o ides he g ea es esis ance
o comp essi e o ces [20–22].
Be ween he bone and he ca ilage, he e is a zone called calci ied ca ilage. This laye is
sepa a ed om he deep ca ilage laye by a bounda y called he idema k ha ep esen s he
mine aliza ion on [
20
,
23
]. Calci ied ca ilage is composed o hype ophic chond ocy es,
and i s main unc ion is o main ain he adhesion o he ca ilage o he bone by ancho ing
he collagen ib ils o he deep zone o he subchond al bone [20,22–24].
Pha maceu ics 2022,14, 1578 4 o 33
Bone issue, known as subchond al bone, is a undamen al issue o he join ’s p ope
unc ionali y, since i abso bs he impac s and p o ides suppo . In addi ion, i dis ibu es
he mechanical loads h oughou he join wi h a g adual ansi ion in s ess and s ain. In
ac , bone is conside ed o be a ha d issue, and i s a e age Young’s modulus anges om 10
o 20 GPa [
25
]. This issue is sepa a ed in o wo zones: subchond al bone pla e and subchond al
bone abeculae. The i s laye is a hin co ical lamella lying immedia ely unde he calci ied
ca ilage. I is composed o channels o ci cula e blood and lympha ic luid om he bone
abeculae o he ca ilage. In con as , he subchond al bone abeculae a e mo e po ous and
me abolically ac i e. They con ain blood essels, ne es, and bone ma ow ha supply he
ca ilage wi h nu ien s and help in i s me abolism [20,23,24].
Apa om os eochond al issue, he join uni is also composed o he syno ial
memb ane and syno ial luid, which a e in ol ed in he pa hogenesis o OA. The syno ial
memb ane is a hin, non-a icula laye composed o wo cell ypes: (i) mac ophages as
a pa o he immune sys em in he join , and (ii) ib oblas s ha sec e e syno ial luid.
Syno ial luid ac s as a lub ican o he a icula su ace, and anspo s nu ien s o he
ca ilage [17,20,24].
1.3. OA
Pa hogenesis and Symp oma ology
In OA, s uc u al al e a ions in he a icula ca ilage and subchond al bone a e ound.
As Figu e 3shows, ca ilage loses i s in eg i y and, hus, is mo e exposed o dis up ion
om physical o ces. To coun e ac ca ilage e osion, chond ocy es inc ease he sec e ion o
molecules ha cause ma ix deg ada ion and p o-in lamma o y media o s. On he o he
hand, bone u no e is inc eased, de eloping bone ma ow lesions. In addi ion, he e is
a ascula in asion h oughou he a ea, and he syno ial memb ane hype ophies and
mac ophages a e ac i a ed, eleasing p o-in lamma o y cy okines. Mo eo e , su ounding
issues, such as ligamen s and pe ia icula muscles, a e o en a ec ed as well [
6
]. OA is
classi ied in o ou di e en s ages, depending on he se e i y g ade and he appea ance o
he symp oms.
Pha maceu ics 2022, 14, x FOR PEER REVIEW 4 o 34
pe pendicula o he su ace, and he GAG con en is he highes . The deep laye p o ides
he g ea es esis ance o comp essi e o ces [20–22].
Be ween he bone and he ca ilage, he e is a zone called calci ied ca ilage. This laye
is sepa a ed om he deep ca ilage laye by a bounda y called he idema k ha ep esen s
he mine aliza ion on [20,23]. Calci ied ca ilage is composed o hype ophic chond o-
cy es, and i s main unc ion is o main ain he adhesion o he ca ilage o he bone by
ancho ing he collagen ib ils o he deep zone o he subchond al bone [20,22–24].
Bone issue, known as subchond al bone, is a undamen al issue o he join ’s
p ope unc ionali y, since i abso bs he impac s and p o ides suppo . In addi ion, i
dis ibu es he mechanical loads h oughou he join wi h a g adual ansi ion in s ess
and s ain. In ac , bone is conside ed o be a ha d issue, and i s a e age Young’s modulus
anges om 10 o 20 GPa [25]. This issue is sepa a ed in o wo zones: subchond al bone
pla e and subchond al bone abeculae. The i s laye is a hin co ical lamella lying immedi-
a ely unde he calci ied ca ilage. I is composed o channels o ci cula e blood and lym-
pha ic luid om he bone abeculae o he ca ilage. In con as , he subchond al bone a-
beculae a e mo e po ous and me abolically ac i e. They con ain blood essels, ne es, and
bone ma ow ha supply he ca ilage wi h nu ien s and help in i s me abolism
[20,23,24].
Apa om os eochond al issue, he join uni is also composed o he syno ial mem-
b ane and syno ial luid, which a e in ol ed in he pa hogenesis o OA. The syno ial
memb ane is a hin, non-a icula laye composed o wo cell ypes: (i) mac ophages as a
pa o he immune sys em in he join , and (ii) ib oblas s ha sec e e syno ial luid. Syn-
o ial luid ac s as a lub ican o he a icula su ace, and anspo s nu ien s o he ca -
ilage [17,20,24].
1.3. OA
Pa hogenesis and Symp oma ology
In OA, s uc u al al e a ions in he a icula ca ilage and subchond al bone a e
ound. As Figu e 3 shows, ca ilage loses i s in eg i y and, hus, is mo e exposed o dis-
up ion om physical o ces. To coun e ac ca ilage e osion, chond ocy es inc ease he
sec e ion o molecules ha cause ma ix deg ada ion and p o-in lamma o y media o s.
On he o he hand, bone u no e is inc eased, de eloping bone ma ow lesions. In addi-
ion, he e is a ascula in asion h oughou he a ea, and he syno ial memb ane hype -
ophies and mac ophages a e ac i a ed, eleasing p o-in lamma o y cy okines. Mo eo-
e , su ounding issues, such as ligamen s and pe ia icula muscles, a e o en a ec ed
as well [6]. OA is classi ied in o ou di e en s ages, depending on he se e i y g ade and
he appea ance o he symp oms.
Figu e 3. Schema ic image o OA’s pa hology and symp oms.
In s age 0, also known as he p e-os eoa h i is s age, he join seems no mal and
heal hy. Ne e heless, cellula damage s a s o occu wi hou any symp oms. S age 1,
o he ea ly s age, is cha ac e ized by he appea ance o bone spu s, and ca ilage begins
o lose i s in eg i y. In his s age he pa ien usually has no symp oms o only mild pain.
Then, in he nex s age—s age 2, o he mild s age— he ca ilage s a s o deg ade due
o enzyme elease. Consequen ly, bone spu s g ow and become pain ul. Join pain and
s i ness commonly appea du ing ac i i ies a his s age. As he disease p og esses, s age
3 o he mode a e s age appea s. He e, ca ilage shows ob ious damage, and he space
be ween he join s becomes na owe . The e o e, pain while mo ing is equen , and join
Pha maceu ics 2022,14, 1578 5 o 33
s i ness wo sens. Finally, s age 4, o se e e OA, occu s. This s age is cha ac e ized by he
p esence o li le ca ilage—o he absence o ca ilage in se e e cases. Syno ial luid is
educed, and bone may e ode, p o oking bone na ow damage. A his s age, signi ican
pain and discom o appea , s i ness and in lamma ion a e se e e, and join dys unc ion
may occu [26–28].
In summa y, a ec ed people expe ience pain and in lamma ion, which a e he mos
common and disabling symp oms. Mo eo e , muscle weakness and join ins abili y a e
equen symp oms. Apa om physical symp oma ology, psychological diso de s due o
pain oge he wi h insomnia and a igue should be aken in o accoun [6,9].
2. Cu en T ea men s
OA is a p og essi e and degene a i e join disease wi h no cu e. T ea men s in he
ea ly s ages o OA a e ocused on gi ing educa ional in o ma ion o pa ien s, weigh loss,
and mode a e physical exe cise, whe eas when he diso de p og esses, cu en ea men
is based on alle ia ing he main disabling symp om— he ch onic pain [
29
]. The apeu ic
guidelines ecommend s a ing wi h opical ea men and mo ing on o o al ea men
when opical d ugs do no elie e he pain. In mo e ad anced s ages o OA, in a-a icula
injec ions a e he ecommended ea men . Finally, when OA is in he se e e s ages and he
a o emen ioned ea men becomes ine ec i e, su ge y may be ecommended. All o hese
ea men s a e summa ized in Table 1.
Table 1. Summa y o he bene i s and side e ec s o he cu en ea men s o OA.
T ea men Posi i e E ec s Side E ec s
Topical ea men - E ec i e and easy o adminis a e
- Gene ally well- ole a ed - Ine ec i e in ad anced s ages o OA
Ace aminophen o pa ace amol - Fi s choice ea men
- Good elie ing pain
- Con o e sy abou he long- e m e ec i eness
- No an i-in lamma o y e ec s
- Hepa o oxic when abused
O al NSAIDs
- Fi s -choice ea men
- Good o elie ing pain and imp o ing
join unc ion
- Gas oin es inal and ca diac issues in he
long- e m and when abused
SYSADOA
- Sa e and well- ole a ed
- Pain elie and imp o emen in join s’
physical unc ion
- Unclea he apeu ic mechanisms
- Disc epancies among he apeu ic guides
In a-a icula injec able HA
- Sa e and well- ole a ed
- An i-in lamma o y e ec s and pain
educ ion
- Bene i s only in he sho - e m pe iod
- Repe i i e in a-a icula injec ions
- Only use ul in mild and mode a e s ages o OA
In a-a icula injec able
co icos e oids
- Good o educing join in lamma ion
and dys unc ion
- Bene i s only in he sho - e m pe iod
- Repe i i e in a-a icula injec ions
- Con o e sial bene i s in knee join s and in he
long- e m
Opia es - Excellen painkille s when o he
ea men s ail
- Tole ance and dependence
- Nega i e bene i / isk a io
- Highly discou aged
Su ge y
- Las he apeu ic op ion
- Rele an imp o emen , especially in
young pa ien s
- Mo e likely o ha e complica ions associa ed wi h
su ge y in he elde ly popula ion
- P obabili y o ejec ion
- Pain is s ill ecu en
PRP - Relie o OA symp oms
- No side e ec s
- Limi ed o knees
- Va iabili y among pa ien s
- Unclea dosage and plasma ex ac ion p o ocols
- E icacy dec eases wi h NSAIDs
Sphe ox™ - Os eochond al egene a ion
- Gene al imp o emen
- No a ailable in all hospi als
- High cos s and long egula o y p ocedu es
- Con aindica ed in ad anced OA
- Only applicable o knee de ec s
Ac onyms—OA: os eoa h i is; NSAIDs: nons e oidal an i-in lamma o y d ugs; SYSADOA: symp oma ic slow-
ac ing d ugs in os eoa h i is; HA: hyalu onic acid; PRP: pla ele - ich plasma.

Pha maceu ics 2022,14, 1578 6 o 33
2.1. Pha macological T ea men s
Topical ea men is based on opical nons e oidal an i-in lamma o y d ugs (NSAIDs).
These demons a e good e ec i eness o pain educ ion, and he side e ec s ha hey
may p oduce a e a e. As hey a e usually well- ole a ed and ha e an easy mode o
adminis a ion, hey a e highly ecommended in he i s s ages o he disease. Ne e heless,
hey become ine ec i e as he disease p og esses and he pain inc eases [6,30,31].
When pain inc eases, o al d ug in ake is ecommended. Among he used d ugs,
ace aminophen o pa ace amol is a well-known d ug o educe mild- o-mode a e pain [
6
,
30
].
I is usually p esc ibed because se e al guidelines ecommend i o OA. Howe e , he e
is an inc easing con o e sy abou i s e icacy in his illness. Me a-analyses ha e e ealed
li le in he way o sa is ac o y e ec s in compa ison wi h placebo [
6
,
30
]. Fu he mo e, he
hepa o oxic side e ec s a e long- e m usage wi h high doses a e a d awback o ake in o
accoun [30]. The e o e, he use o his d ug may be es ic ed o sho - e m pe iods [31].
NSAIDs a e he ea men o choice, since i has been shown ha hey dec ease
pain and imp o e join unc ion [
30
]. Howe e , long- e m ea men s a high doses ha e
conside able side e ec s, such as gas oin es inal issues and neph o oxici y [
30
,
31
]. COX-
2-selec i e inhibi o s a e a o m o NSAIDs ha may a oid hese p oblems, bu a e con-
aindica ed in pa ien s wi h ca dio ascula p oblems [
31
]. As a consequence, hei use
should be es ic ed o sho ea men pe iods, making a good he apeu ic app oach o
ch onic diseases such as OA impossible.
Symp oma ic slow-ac ing d ugs in OA (SYSADOA), such as glucosamine and chon-
d oi in sul a e (CS), a e widely p esc ibed. CS is a GAG na u ally ound in he ECM,
whe eas glucosamine is a me abolic p ecu so o GAGs. The e is much con o e sy in he
li e a u e ega ding he use o hese subs ances. They ha e he ad an age o being sa e and
showing almos no side e ec s, bu hei he apeu ic mechanism is unclea , and while some
me a-analyses indica e hei po en ial bene i s in pain elie and imp o emen in physical
join unc ion, o he s s ongly discou age hei use [30,31].
Fo pa ien s who do no espond well o o al ea men s, in a-a icula injec ions o
hyalu onic acid (HA) a e ecommended. HA is a molecule om he g oup o GAGs ha a e
na u ally ound in he join s’ syno ial luid. I s main unc ion is he lub ica ion o he join s.
I has also been epo ed o be chond op o ec i e agains mechanical damage. I has been
ound ha in a-a icula injec ions o HA educe pain, ha e an i-in lamma o y e ec s, and
p omo e GAG syn hesis. I is gene ally sa e and e ec i e in mild- o-mode a e s ages o
knee OA. As a d awback, i s long- e m e ec s a e limi ed, and epe i i e injec ions o HA
a e usually uncom o able o pa ien s [30,31].
In addi ion, in a-a icula co icos e oids a e e y common d ugs used o ea
in lamma o y- ela ed diseases; he e o e, hey a e expec ed o be bene icial o ea OA as
well, by educing join in lamma ion, pain, and dys unc ion. All o he clinical e idence
has demons a ed pain educ ion a e co icos e oid injec ions. Ne e heless, his bene i
has only been obse ed in sho - e m pe iods, and epe i i e injec ions ha e no been
associa ed wi h long- e m pain educ ion. In addi ion, as he s udies ha e ocused on knee
join ea men , whe he hese d ugs a e bene icial o o he join s is unclea . Fu he mo e,
a ecen me a-analysis showed li le imp o emen in join unc ion, and epo ed g ea e
ca ilage damage han in he placebo g oup a e 2 yea s o co icos e oid adminis a ion.
Thus, he use o his kind o d ug o OA ea men has become con o e sial [6,30].
As an al e na i e, some guidelines ecommend he use o opia es. Al hough hey
a e qui e e ec i e in elie ing pain, he side e ec s ha hey p oduce a e ex ensi e and
se ious, such as ole ance and dependence. In ac , opioid abuse has been ecognized as an
epidemic in he US, whe e g ea e o s a e being made in o de o educe hei use [
32
].
Apa om his se ious p oblem, opioids ha e shown only a small imp o emen in he
OA symp oma ology, wi h an inc ease in side e ec s a e opia e adminis a ion. Thus, he
use o opia es is highly discou aged, and hey should only be p esc ibed o sho - e m
ea men s and when o he he apeu ic op ions a e no possible [6,30,31].
Pha maceu ics 2022,14, 1578 7 o 33
2.2. Su ge y
Di e en su gical in e en ions ha e been ca ied ou in he clinics. A bone ma ow
s imula ion echnique known as mic o ac u e is ecommended o ea small chond al
de ec s (less han 2 cm
2
). When he disease eaches he subchond al bone, os eochond al
au og a ansplan a ion is he sugges ed op ion. This su gical p ocedu e is di ided in o
mosaicplas y, which is based on ansplan ing mul iple small, ci cula os eochond al g a s,
and he single-plug echnique, which consis s o implan ing a single, la ge g a . Excellen
esul s ha e been epo ed a e he implemen a ion o bo h echniques, bu he e a e s ill
disad an ages in e ms o dono si e mo bidi y and pa ien age limi a ion, as he p ocedu e
is es ic ed o pa ien s o e 50 yea s old [
33
]. In o de o ea g ea e os eochond al de ec s,
allog a implan a ion has been sugges ed, bu he e may be limi a ions in acqui ing he
g a due o dono una ailabili y [34].
To al join eplacemen su ge y o a h oplas y is he las he apeu ic op ion. Du ing
his su ge y, he damaged join is eplaced wi h an a i icial implan ha is made o me al,
ce amic, o plas ic [
33
]. This is ecommended o pa ien s wi h se e e OA whose quali y o
li e is conside ably educed. Clinically ele an imp o emen s ha e been obse ed, bu
complica ions associa ed wi h su ge y in he elde ly popula ion a e common. In ec ions,
neu o ascula inju y, and pe i-implan ac u es a e he main complica ions. Addi ionally,
implan ejec ions a e a ound 12%, and pain is s ill a ecu en symp om [6,30].
2.3. Biological The apies
Recen ly, apa om adi ional d ugs and su ge y, ea men s based on in a-a icula
injec ion o pla ele - ich plasma (PRP) ha e a ac ed signi ican clinical in e es . This he -
apy consis s o inocula ing au ologous plasma on he join , because i has been shown ha
he PRP eleases bioac i e molecules (e.g., g ow h ac o s, cy okines, o an i-in lamma o y
media o s). This has been epo ed o elie e OA symp oms and o demons a e no side
e ec s. Ne e heless, PRP is limi ed o he knee join , and a iabili y has been obse ed
among pa ien s. This can be explained by he absence o a clea dosage guide and he lack
o a s anda dized plasma ex ac ion p o ocol. Mo eo e , simul aneous ea men wi h PRP
and NSAIDs educes he PRP’s e icacy [35,36].
Au ologous chond ocy e implan a ion (ACI) has also been used o he ea men o
os eochond al de ec s o yea s. This ea men , au ho ized by he Eu opean Medicines
Agency (EMA) as an ad anced he apy medicinal p oduc , is based on he implan a ion o
au ologous chond ocy es in he join o p omo e i s egene a ion and, he e o e, alle ia e
he symp oms. In ac , Sphe ox
™
is he only ACI p oduc ha has been comme cialized.
This he apy is based on he implan a ion o sphe ical agg ega es ha a e composed o
au ologous human chond ocy es expanded ex i o and an au o-syn he ized ex acellula
ma ix. Howe e , his ea men has also shown some d awbacks, such as he necessi y o
expe su geons o i s applica ion, as well as he au ho iza ion o he hospi al in which he
he apy is applied. Mo eo e , ex i o cell expansion equi es s ong egula o y p ocedu es.
Consequen ly, his ea men is no a ailable in all hospi als, and i has high cos s. In
addi ion, i is con aindica ed in ad anced OA s ages (i.e., s ages 3 and 4), and i is only
p esc ibed o knee join de ec s [37].
In conclusion, cu en ea men s can alle ia e he symp oms p oduced by OA in
he sho - e m. In he long- e m, as he disease p og esses and he pain becomes in ense,
cu en ea men s ail o imp o e he pa ien ’s quali y o li e. In addi ion, gi en ha he
a ge popula ion ha su e s om OA is he elde ly, he appea ance o o he diseases
ha could hinde he gene al use o hese d ugs should be highligh ed. The e o e, new
he apeu ic app oaches should be p oposed.
3. New The apeu ic App oaches: Tissue Enginee ing and 3D Biop in ing
As men ioned abo e, one o he mos in e es ing he apeu ic app oaches o OA is he
use o ACI. This kind o cell he apy has been widely esea ched and imp o ed, since i
can p o ec ca ilage om deg ada ion and, consequen ly, cause emission o he disease’s
Pha maceu ics 2022,14, 1578 8 o 33
symp oma ology. In ac , he e a e se e al s udies, including clinical ials, in which his
he apy has shown p omising esul s [
37
]. Ano he app oach ha is gaining a en ion in
cell he apy ea men s is he use o mesenchymal s omal cells (MSCs), as i has been
epo ed ha he a icula adminis a ion o MSCs in he knee elie es pain and imp o es
i s unc ion [
38
]. Likewise, adipose-de i ed s omal cells (ASCs) ha e also been applied
o chond al egene a ion, since hey can be ha es ed wi h educed mobili y a he dono
si e in compa ison wi h o he MSC sou ces [
39
]. Fu he mo e, bo h ASCs and MSCs
ha e he po en ial o sec e e an i-in lamma o y and immunomodula o y molecules, which
complemen hei adminis a ion as an OA ea men [
40
,
41
]. Howe e , he long- e m
bene i s a e con o e sial. In addi ion, i has been p o en ha injec ion o MSCs h ough a
needle comp omises hei iabili y due o shea o ces and ha , a e adminis a ion, cells
end o mig a e, making i di icul o sec e e he apeu ically ac i e molecules [
40
]. The
implemen a ion o a cellula suppo would no only a oid hese d awbacks, bu also ake
in o accoun he mechanical p ope ies ha a e o g ea impo ance in he egene a ion
o he join . In ac , hyd ogel-based cellula suppo s ha e been al eady s udied wi h
success ul esul s in e ms o mimicking na i e mechanical p ope ies and imp o ing cell
iabili y [42,43].
In his con ex , issue enginee ing, which b ings oge he cell he apy, bioma e ial
enginee ing, and he deli e y o d ugs o he apeu ic molecules, has become he mos
p omising he apeu ic app oach (Figu e 4) [
16
,
44
,
45
]. I is based on he manu ac u e o
h ee-dimensional (3D) s uc u es o sca olds ha suppo cells, allowing hem o adhe e,
p oli e a e, and di e en ia e. These s uc u es can also con ain di e en elemen s, such as
d ugs, g ow h ac o s, and he apeu ic molecules [16].
Pha maceu ics 2022, 14, x FOR PEER REVIEW 9 o 34
Figu e 4. (A) Diag am o he elemen s used in issue enginee ing. Adap ed om [46]. (B) Scheme o
di e en biop in ing me hods. Adap ed om [47].
Among sca old manu ac u ing echniques, 3D biop in ing has gained signi icance
in ecen yea s. This addi i e manu ac u ing echnique is cha ac e ized by he ab ica ion
o laye -by-laye s uc u es ia compu e -aided design (CAD). CAD gene a es a G-code
ha can be ead by he biop in e [48]. The c ea ion o he design in CAD as well as he
modi ica ion o he G-code allows he o al con ol o he shape and s uc u e o he sca -
old, g an ing biop in ing an ad an age o e adi ional manu ac u ing echniques. Fu -
he mo e, biop in ing echniques pe mi he addi ion o high cell densi ies, while o he
echniques a e unable o do so, o he cells ha e o be added a e making he sca old [49].
In ac , ana omically speci ic implan s could be designed o each pa ien using his ech-
nology. Ano he challenge in he issue enginee ing ield o join egene a ion is he ac
ha he join is made up o wo issues—ca ilage and bone—which, in u n, ha e sepa a e
zones wi h di e en cell densi ies, composi ions, and biomechanics. Th ee-dimensional
biop in ing, as an addi i e echnique, allows he manu ac u ing o sca olds wi h di e en
laye s; he e o e, he na i e issue can be imi a ed. In addi ion, i is as and au oma ic,
and accep s a wide a ie y o ma e ials, wmaking i a p omising echnique in his ield
[48,50].
The deposi ed ma e ial is known as bio-ink. These bio-inks a e composed o cells and
bioma e ials o which o he molecules such as d ugs, p o eins, gene ic ma e ial, o g ow h
ac o s may be added [51]. Howe e , he bioma e ial, whe he o na u al o syn he ic
o igin, has o mee ce ain equi emen s o be conside ed a bio-ink [50]. In he i s place,
i has o be biocompa ible wi h he cells, since i has o suppo cell a achmen , mig a ion,
p oli e a ion, and di e en ia ion. Second, i has o be biodeg adable and, inally, i has o
be p in able, which necessi a es aking in o accoun i s heological p ope ies and gela ion
Figu e 4.
(
A
) Diag am o he elemen s used in issue enginee ing. Adap ed om [
46
]. (
B
) Scheme o
di e en biop in ing me hods. Adap ed om [47].
Among sca old manu ac u ing echniques, 3D biop in ing has gained signi icance
in ecen yea s. This addi i e manu ac u ing echnique is cha ac e ized by he ab ica ion
Pha maceu ics 2022,14, 1578 9 o 33
o laye -by-laye s uc u es ia compu e -aided design (CAD). CAD gene a es a G-code
ha can be ead by he biop in e [
48
]. The c ea ion o he design in CAD as well as he
modi ica ion o he G-code allows he o al con ol o he shape and s uc u e o he sca old,
g an ing biop in ing an ad an age o e adi ional manu ac u ing echniques. Fu he mo e,
biop in ing echniques pe mi he addi ion o high cell densi ies, while o he echniques
a e unable o do so, o he cells ha e o be added a e making he sca old [
49
]. In ac ,
ana omically speci ic implan s could be designed o each pa ien using his echnology.
Ano he challenge in he issue enginee ing ield o join egene a ion is he ac ha
he join is made up o wo issues—ca ilage and bone—which, in u n, ha e sepa a e
zones wi h di e en cell densi ies, composi ions, and biomechanics. Th ee-dimensional
biop in ing, as an addi i e echnique, allows he manu ac u ing o sca olds wi h di e en
laye s; he e o e, he na i e issue can be imi a ed. In addi ion, i is as and au oma ic, and
accep s a wide a ie y o ma e ials, wmaking i a p omising echnique in his ield [48,50].
The deposi ed ma e ial is known as bio-ink. These bio-inks a e composed o cells and
bioma e ials o which o he molecules such as d ugs, p o eins, gene ic ma e ial, o g ow h
ac o s may be added [
51
]. Howe e , he bioma e ial, whe he o na u al o syn he ic o igin,
has o mee ce ain equi emen s o be conside ed a bio-ink [
50
]. In he i s place, i has o be
biocompa ible wi h he cells, since i has o suppo cell a achmen , mig a ion, p oli e a ion,
and di e en ia ion. Second, i has o be biodeg adable and, inally, i has o be p in able,
which necessi a es aking in o accoun i s heological p ope ies and gela ion kine ics.
Mo eo e , he bioma e ial mus ha e p ope mechanical p ope ies and be bioac i e [52].
The e a e di e en 3D biop in ing echniques, including ex usion-based, inkje -based,
and lase -assis ed biop in ing (Figu e 4B). Each o hese echniques is based on di e en
p inciples. Ex usion-based biop in ing is he mos common echnique, since i is easy o use,
economical, and lexible in he use o a wide ange o ma e ials [
53
]. I is based on he
con inuous deposi ion o he bio-ink in a ilamen o m h ough a needle ia he applica ion
o mechanical p essu e o ai p essu e (pneuma ic). Inkje -based biop in ing is cha ac e ized
by he deposi ion o he bio-ink in a d ople o m a e he applica ion o a piezoelec ic o
elec os a ic d op-on-demand sou ce. Lase -assis ed biop in ing uses a lase ene gy beam o
he deposi ion o he bio-ink [53,54].
Se e al s udies ha e implemen ed biop in ing echniques o join egene a ion. The
majo i y o hose s udies ocus on one o he wo issues in ol ed—ca ilage o bone—whe eas
ewe s udies a e based on he de elopmen o he whole os eochond al uni by 3D biop in ing.
3.1. 3D Biop in ing in Ca ilage
Ca ilage issue lacks blood essels, ne es, and a lympha ic sys em, making his issue
an ideal a ge o 3D biop in ing in compa ison wi h o he , mo e complex issues [
24
,
44
].
Ne e heless, ca ilage is subjec ed o high shea o ces. Thus, he challenge, when i comes
o biop in ing ca ilage, is o mee he equi emen s in e ms o mechanical p ope ies, as
well as o mimic he laye ed s uc u e o he na i e issue as closely as possible.
To ob ain 3D s uc u es esis an o mechanical p essu es, some s udies ha e ocused
on seeding cells on p e iously 3D-p in ed sca olds, using he moplas ic polyme s such as
polycap olac one (PCL) o polylac ic acid (PLA) [
55
]. Elec ospinning, which enables he
ab ica ion o polyme ic ibe s, is ano he inno a i e echnology ha has been used o his
pu pose, since nano ibe s ein o ce he sca old [56]. Howe e , 3D biop in ing echnology
equi es he inclusion o he biological pa (li ing cells) in he bio-ink, so he use o hese
ma e ials is unsui able o cells, as hese polyme s need high empe a u es o be ex uded.
Fo his eason, hyd ogels a e he mos used op ion, as hey ha e he abili y o abso b wa e ,
a e biocompa ible wi h he cells, and a e biodeg adable.
Di e en bioma e ials ha e been s udied o de elop bio-inks. Among hem, bioma e i-
als ha a e na u ally ound in os eochond al issue—such as collagens o GAGs—ha e been
p oposed, as along wi h o he s ha ha e g ea e p in abili y cha ac e is ics o mechanical
esis ance, such as algina e, gela in, o silk ib oin. All o hese s udies a e ep esen ed in
Table 2.
Pha maceu ics 2022,14, 1578 16 o 33
Zhang e al. [
62
] p oposed mixing decellula ized goa ca ilage ECM wi h silk ib oins.
They included abbi bone ma ow MSCs, TGF-
β
3 as a chond ogenic g ow h ac o , and
polye hylene glycol (PEG) 400 as a c osslinke . A e a heological s udy o de e mine he
op imal concen a ions o he bio-ink, hey managed o manu ac u e po ous sca olds ia
ex usion. The cell iabili y, p oli e a ion, and chond ogenic di e en ia ion we e good, and
we e p opo ional o he amoun o dECM. Fu he mo e, hey ob ained a sus ained elease
o TGF-
β
3 om he sca old, p omo ing he p oduc ion o collagen and GAGs. Finally,
biop in ed sca olds we e implan ed subcu aneously in mice.
In i o
esul s showed an
inc ease in he p oduc ion o GAGs and collagen, as well as in he sca olds’ mechan-
ical p ope ies, making hese sca olds a p omising he apeu ic app oach o ca ilage
egene a ion [62].
In a p e ious wo k by he same esea ch g oup, Li e al. [
63
] de eloped a bio-ink by
combining silk ib oin, abbi pla ele - ich plasma (PRP), abbi chond ocy es, and PEG
400. A e ex usion biop in ing, hey showed ha he addi ion o plasma inc eased he
sca olds’ mechanical p ope ies as well as cell iabili y and p oli e a ion. In e es ingly,
he plasma con ained a ious g ow h ac o s ha we e deli e ed om he sca old o
p omo e cell unc ionali y and chond ogenic di e en ia ion [
63
]. In ac , silk ib oin has
gained popula i y as a bio-ink componen because i is biocompa ible, biodeg adable,
and has ema kable mechanical s eng h [
62
]. Ano he wo k using silk ib oin as a bio-ink
componen was he one p oposed by Singh e al [
64
]., who de eloped a bio-ink based on wo
ypes o silk ib oin, gela in, and po cine p ima y chond ocy es. They ob ained po ous and
p in able sca olds ia ex usion-based biop in ing (Figu e 7). Cells showed high iabili y
and p oli e a ion abili y inside he sca olds, as well as chond ogenic gene exp ession and
ca ilage ECM p oduc ion. Mo eo e , he au ho s injec ed he bio-ink subcu aneously in
mice o s udy hei immune esponse. As a esul , a long immune esponse was no ound,
so hey sugges ed he use o hese sca olds o ca ilage egene a ion. Ne e heless, he
sca old mechanical p ope ies we e lowe (143 kPa) han hose o na i e ca ilage; he e o e,
he sca olds would only be bene icial o so issue egene a ion [64].
Pha maceu ics 2022, 14, x FOR PEER REVIEW 16 o 34
ex usion-based biop in ing. Cell iabili y and p oli e a ion we e inc eased p opo ion-
ally o he dECM concen a ion. dECM also p omo ed cells o p oduce GAGs and colla-
gen. In addi ion, he sca old mechanical p ope ies we e also posi i ely imp o ed by he
inclusion o dECM [61]. These p omising esul s a e including dECM ha e also been
epo ed by o he esea che s. Zhang e al. [62] p oposed mixing decellula ized goa ca -
ilage ECM wi h silk ib oins. They included abbi bone ma ow MSCs, TGF-β3 as a chon-
d ogenic g ow h ac o , and polye hylene glycol (PEG) 400 as a c osslinke . A e a heo-
logical s udy o de e mine he op imal concen a ions o he bio-ink, hey managed o
manu ac u e po ous sca olds ia ex usion. The cell iabili y, p oli e a ion, and chond o-
genic di e en ia ion we e good, and we e p opo ional o he amoun o dECM. Fu he -
mo e, hey ob ained a sus ained elease o TGF-β3 om he sca old, p omo ing he p o-
duc ion o collagen and GAGs. Finally, biop in ed sca olds we e implan ed subcu ane-
ously in mice. In i o esul s showed an inc ease in he p oduc ion o GAGs and collagen,
as well as in he sca olds’ mechanical p ope ies, making hese sca olds a p omising he -
apeu ic app oach o ca ilage egene a ion [62].
In a p e ious wo k by he same esea ch g oup, Li e al. [63] de eloped a bio-ink by
combining silk ib oin, abbi pla ele - ich plasma (PRP), abbi chond ocy es, and PEG
400. A e ex usion biop in ing, hey showed ha he addi ion o plasma inc eased he
sca olds’ mechanical p ope ies as well as cell iabili y and p oli e a ion. In e es ingly,
he plasma con ained a ious g ow h ac o s ha we e deli e ed om he sca old o p o-
mo e cell unc ionali y and chond ogenic di e en ia ion [63]. In ac , silk ib oin has
gained popula i y as a bio-ink componen because i is biocompa ible, biodeg adable, and
has ema kable mechanical s eng h [62]. Ano he wo k using silk ib oin as a bio-ink
componen was he one p oposed by Singh e al [64]., who de eloped a bio-ink based on
wo ypes o silk ib oin, gela in, and po cine p ima y chond ocy es. They ob ained po-
ous and p in able sca olds ia ex usion-based biop in ing (Figu e 7). Cells showed high
iabili y and p oli e a ion abili y inside he sca olds, as well as chond ogenic gene ex-
p ession and ca ilage ECM p oduc ion. Mo eo e , he au ho s injec ed he bio-ink sub-
cu aneously in mice o s udy hei immune esponse. As a esul , a long immune esponse
was no ound, so hey sugges ed he use o hese sca olds o ca ilage egene a ion.
Ne e heless, he sca old mechanical p ope ies we e lowe (143 kPa) han hose o na i e
ca ilage; he e o e, he sca olds would only be bene icial o so issue egene a ion [64].
Figu e 7. Silk ib oin (SF)-based sca olds. Ex usion biop in ing p ocess o SF + gela in bio-ink, ob-
aining po ous sca olds. Scale ba = 200 µm. Adap ed om [64].
Achie ing mechanical p ope ies in he sca olds ha a e simila o hose o human
ca ilage is challenging when using hyd ogels such as algina e, silk ib oin, o collagen by
Figu e 7.
Silk ib oin (SF)-based sca olds. Ex usion biop in ing p ocess o SF + gela in bio-ink,
ob aining po ous sca olds. Scale ba = 200 µm. Adap ed om [64].
Achie ing mechanical p ope ies in he sca olds ha a e simila o hose o human
ca ilage is challenging when using hyd ogels such as algina e, silk ib oin, o collagen by
hemsel es. Consequen ly, o he bio-ink componen s ha e been p oposed. Fo example,
Li e al. [
65
] de eloped a bio-ink con aining chemically modi ied chi osan o make i
soluble, along wi h chond oi in sul a e, since i has been epo ed o be in ol ed in he
mechanical esponse o na i e ca ilage as well as in ca ilage egene a ion [
73
]. They
included human adipose-de i ed MSCs and used plu onic as sac i icial ink o gi e suppo
o he sca old. Once hey manu ac u ed he sca olds by ex usion, hey s udied hei

Pha maceu ics 2022,14, 1578 17 o 33
biocompa ibili y in mice by subcu aneous implan a ion. Immune esponse dec eased
wi hin he ollowing days a e implan a ion. In e es ingly, ca ilage-deg ada i e cy okines
we e educed as a consequence o he chond oi in’s an i-in lamma o y e ec s, sugges ing
he use o chond oi in o ca ilage egene a ion pu poses [
65
]. Howe e , he sca olds’
mechanical p ope ies and how he chond oi in was in ol ed in hem we e no shown.
Simila o chond oi in sul a e, HA has been epo ed o ha e p omising p ope ies o
egene a ing ca ilage. Howe e , i has poo heological p ope ies o use as a bio-ink wi h-
ou any o he suppo i e componen (s). Gala aga e al. [
66
] modi ied hyalu onic acid o
c ea e no bo nene-modi ied HA, which was c osslinkable wi h isible ligh . In addi ion, hey
de eloped an in si u c osslinking echnique ha consis ed o exposing he bio-ink o isible
ligh jus a e being ex uded (Figu e 8A). Thus, hey de eloped HA sca olds wi hou he
addi ion o any heological componen o he bio-ink. Bo ine bone ma ow MSCs we e iable
a e biop in ing, indica ing ha he echnique was biocompa ible. Mo eo e , cells’ chon-
d ogenic di e en ia ion and sca old mechanical p ope ies inc eased a e biop in ing [
66
].
O he esea ch g oups ha e also de eloped in si u biop in ing echniques. In he case o
Di Bella e al [
67
]., hey used an ex usion-based handled biop in ing echnique based on a
coaxial sys em called “Biopen” (Figu e 8A). The bio-ink was made o o ine adipose-de i ed
MSCs, hyalu onic me hac yla e, and gela in me hac yla e. The au ho s demons a ed high
cell iabili y a e using his echnique in a p e ious wo k [
74
], and his s udy was ocused on
in i o
esea ch using a chond al de ec sheep model. They compa ed he use o he Biopen
wi h sca olds biop in ed using a con en ional biop in e . As a esul , no di e ences we e
obse ed in e ms o ca ilage egene a ion and mechanical p ope ies be ween he sca olds
c ea ed by he Biopen and he con en ional biop in e ; in ac , hey we e good in bo h cases.
Ne e heless, Biopen- ab ica ed sca olds showed be e o e all mac oscopic and mic oscopic
cha ac e is ics, oge he wi h excellen applicabili y and handling o he echnique by he
su geons. As a poin o imp o emen , bo h implan s ailed o adhe e o he hos issue.
The e o e, chemical modi ica ions o he bio-ink could be needed [67].
Pha maceu ics 2022, 14, x FOR PEER REVIEW 18 o 34
Figu e 8. In si u biop in ing echniques: (A) In si u c osslinking echnique consis ing o exposing
he bio-ink o isible ligh jus a e being ex uded. Adap ed om [66]. (B) “Biopen”—ex usion-
based handled biop in ing echnique based on a coaxial sys em. Adap ed om [67].
Gela in me hac yla e (GelMA) has also been widely applied in ca ilage egene a ion
ia 3D biop in ing due o i s desi able as c osslinking using UV ligh , oge he wi h i s
biodeg adabili y, biocompa ibili y, and limi ed an igenici y. Ruiz-Can u e al. [68] used
ex usion-based biop in ing o manu ac u e po ous sca olds using a GelMA bio-ink wi h
o ine chond ocy es. GelMA, as a cell ca ie , p o ed o be good in e ms o high cell ia-
bili y and p oli e a ion a e biop in ing. In addi ion, cells also managed o p oduce GAGs
and collagen. Despi e he ac ha mechanical p ope ies inc eased a e biop in ing, hey
did no achie e na i e ca ilage alues. Consequen ly, he au ho s p oposed he addi ion
o PCL h ough 3D p in ing as a mechanical suppo . The hyb id GelMA–PCL sca old
showed he same abili y o main ain good cell iabili y (Figu e 9A) and chond ogenic
unc ionali y a e biop in ing. In e es ingly, he mechanical p ope ies s ongly in-
c eased, sugges ing he use o hyb id sca olds as a ca ilage egene a ion s a egy [68]. A
simila app oach was pe o med by de Ruij e e al. [69] In his case, he sca old bi-
op in ed by ex usion, and composed o GelMA and equine MSCs, was ein o ced wi h
PCL ibe s using a mel elec ow i ing echnique. This elec ospinning echnology uses a
high- ol age elec ical ield o o m sub-mic ome e ibe s om polyme mel s. The au-
ho s showed ha he inclusion o his echnique had no nega i e e ec s on cell iabili y
and p oli e a ion. In ac , cells main ained he abili y o p oduce GAGs and collagen a e
biop in ing. The e o e, as Figu e 9B shows, he inclusion o PCL wi h his echnique may
be a good op ion o inc ease he mechanical p ope ies o sca olds o ca ilage egene a-
ion [69].
Figu e 8.
In si u biop in ing echniques: (
A
) In si u c osslinking echnique consis ing o exposing he
bio-ink o isible ligh jus a e being ex uded. Adap ed om [
66
]. (
B
) “Biopen”—ex usion-based
handled biop in ing echnique based on a coaxial sys em. Adap ed om [67].
Pha maceu ics 2022,14, 1578 18 o 33
Gela in me hac yla e (GelMA) has also been widely applied in ca ilage egene a ion
ia 3D biop in ing due o i s desi able as c osslinking using UV ligh , oge he wi h i s
biodeg adabili y, biocompa ibili y, and limi ed an igenici y. Ruiz-Can u e al. [
68
] used
ex usion-based biop in ing o manu ac u e po ous sca olds using a GelMA bio-ink wi h
o ine chond ocy es. GelMA, as a cell ca ie , p o ed o be good in e ms o high cell iabili y
and p oli e a ion a e biop in ing. In addi ion, cells also managed o p oduce GAGs and
collagen. Despi e he ac ha mechanical p ope ies inc eased a e biop in ing, hey did
no achie e na i e ca ilage alues. Consequen ly, he au ho s p oposed he addi ion o PCL
h ough 3D p in ing as a mechanical suppo . The hyb id GelMA–PCL sca old showed
he same abili y o main ain good cell iabili y (Figu e 9A) and chond ogenic unc ionali y
a e biop in ing. In e es ingly, he mechanical p ope ies s ongly inc eased, sugges ing
he use o hyb id sca olds as a ca ilage egene a ion s a egy [
68
]. A simila app oach
was pe o med by de Ruij e e al. [
69
] In his case, he sca old biop in ed by ex usion,
and composed o GelMA and equine MSCs, was ein o ced wi h PCL ibe s using a mel
elec ow i ing echnique. This elec ospinning echnology uses a high- ol age elec ical
ield o o m sub-mic ome e ibe s om polyme mel s. The au ho s showed ha he
inclusion o his echnique had no nega i e e ec s on cell iabili y and p oli e a ion. In ac ,
cells main ained he abili y o p oduce GAGs and collagen a e biop in ing. The e o e, as
Figu e 9B shows, he inclusion o PCL wi h his echnique may be a good op ion o inc ease
he mechanical p ope ies o sca olds o ca ilage egene a ion [69].
Pha maceu ics 2022, 14, x FOR PEER REVIEW 19 o 34
Figu e 9. GelMA-based sca old: (A) Rep esen a i e b igh - ield and luo escence images o
hyb id sca olds composed o PCL and GelMA. Scale ba = 2 mm. Adap ed om [68]. (B) Sche-
ma ic image o ex usion-based biop in ing and elec ow i ing echniques ha imp o ed sca -
old mechanical p ope ies * = p < 0.05. Adap ed om [69].
Zhu e al. [70] p oposed ano he biop in ing app oach using GelMA/human bone
ma ow MSCs bio-ink. Fi s , hey included polye hylene glycol diac yla e (PEGDA) in
o de o inc ease he mechanical p ope ies. Then, hey added he g ow h ac o TFG-β1
encapsula ed in poly-lac ic-co-glycolic acid (PLGA) o p omo e chond ogenic di e en ia-
ion. Finally, hey used s e eoli hog aphy-based 3D biop in ing, which is a lase -assis ed
biop in ing me hod o manu ac u e sca olds. As a esul , hey showed ha PEGDA im-
p o ed mechanical p ope ies and p in abili y. Impo an ly, cell iabili y and p oli e a-
ion we e high despi e he c osslinking p ocedu e wi h UV ligh . Mo eo e , a sus ained
elease o TFG-β1 om he sca old was ound, which enhanced cells’ chond ogenic phe-
no ype exp ession a e biop in ing [70]. Consequen ly, simila o ex usion-based bi-
op in ing, wi h his echnique, i is possible o ob ain adequa e sca olds o ca ilage e-
gene a ion pu poses.
Apa om mechanics, he o he challenge when i comes o biop in ing s uc u es
in o de o subs i u e damaged ca ilage is he c ea ion o sca olds ha simula e he in-
e nal laye ed s uc u e o he na i e ca ilage. To do so, Wu e al. [71] combined ex usion
biop in ing wi h aspi a ion-assis ed biop in ing (AAB), which allows p ecise posi ioning
o sphe oids by employing aspi a ion o li indi idual sphe oids and biop in hem on o
a hyd ogel. By using sodium algina e and human adipose-de i ed s em cells (ADSCs)
hey manu ac u ed a wo-laye ed sca old simula ing he deep and supe icial laye s o
he ca ilage. As Figu e 10A shows, i s , hey de eloped he deepe laye h ough AAB
by deposi ing sphe oids e ically wi h he suppo o a pin ay. Then, he supe icial laye
was ex uded ho izon ally on he o he laye . The esul ing sca old showed high cell i-
abili y a e bo h biop in ing echniques, as well as mechanical p ope ies simila o hose
o he na i e ca ilage (2.1 MPa). In e es ingly, cells deposi ed collagen ibe s aligned sim-
ila ly o na i e ca ilage [71].
Figu e 9.
GelMA-based sca old: (
A
) Rep esen a i e b igh - ield and luo escence images o hyb id
sca olds composed o PCL and GelMA. Scale ba = 2 mm. Adap ed om [68]. (B) Schema ic image
o ex usion-based biop in ing and elec ow i ing echniques ha imp o ed sca old mechanical
p ope ies * = p< 0.05. Adap ed om [69].
Zhu e al. [
70
] p oposed ano he biop in ing app oach using GelMA/human bone
ma ow MSCs bio-ink. Fi s , hey included polye hylene glycol diac yla e (PEGDA) in
o de o inc ease he mechanical p ope ies. Then, hey added he g ow h ac o TFG-
β
1
encapsula ed in poly-lac ic-co-glycolic acid (PLGA) o p omo e chond ogenic di e en ia-
ion. Finally, hey used s e eoli hog aphy-based 3D biop in ing, which is a lase -assis ed
biop in ing me hod o manu ac u e sca olds. As a esul , hey showed ha PEGDA im-
p o ed mechanical p ope ies and p in abili y. Impo an ly, cell iabili y and p oli e a ion
we e high despi e he c osslinking p ocedu e wi h UV ligh . Mo eo e , a sus ained elease
o TFG-
β
1 om he sca old was ound, which enhanced cells’ chond ogenic pheno ype
exp ession a e biop in ing [
70
]. Consequen ly, simila o ex usion-based biop in ing,
wi h his echnique, i is possible o ob ain adequa e sca olds o ca ilage egene a ion
pu poses.
Pha maceu ics 2022,14, 1578 19 o 33
Apa om mechanics, he o he challenge when i comes o biop in ing s uc u es in
o de o subs i u e damaged ca ilage is he c ea ion o sca olds ha simula e he in e nal
laye ed s uc u e o he na i e ca ilage. To do so, Wu e al. [
71
] combined ex usion
biop in ing wi h aspi a ion-assis ed biop in ing (AAB), which allows p ecise posi ioning
o sphe oids by employing aspi a ion o li indi idual sphe oids and biop in hem on o
a hyd ogel. By using sodium algina e and human adipose-de i ed s em cells (ADSCs)
hey manu ac u ed a wo-laye ed sca old simula ing he deep and supe icial laye s o he
ca ilage. As Figu e 10A shows, i s , hey de eloped he deepe laye h ough AAB by
deposi ing sphe oids e ically wi h he suppo o a pin ay. Then, he supe icial laye was
ex uded ho izon ally on he o he laye . The esul ing sca old showed high cell iabili y
a e bo h biop in ing echniques, as well as mechanical p ope ies simila o hose o he
na i e ca ilage (2.1 MPa). In e es ingly, cells deposi ed collagen ibe s aligned simila ly o
na i e ca ilage [71].
Pha maceu ics 2022, 14, x FOR PEER REVIEW 20 o 34
Figu e 10. Laye ed sca olds: (A) Schema ic image o he manu ac u e o zonally s a i ied a icula
ca ilage. Adap ed om [71]. (B). His ological images o GAGs (sa anin-O, op), collagen ype II
(middle), and collagen ype I (bo om) ma ix o APCs and MSCs in GelMA/gellan gum/HAMA
(GGH) biop in ed sca olds a day 42. Scale ba = 100 μm. Adap ed om [72].
In ano he ecen wo k, Mouse e al. [72] aimed o de elop he e ocellula ca ilage
cons uc s by using h ee di e en cell ypes: equine chond ocy es, MSCs, and a icula
ca ilage p ogeni o cells (ACPC) which we e epo ed o be in he supe icial laye o he
ca ilage. To achie e his, cells we e embedded in o wo inks composed o GelMA/gellan
gum and GelMA/gellan gun/hyalu onic me hac yla e (HAMA). Then, he au ho s c ea ed
sca olds using ex usion-based biop in ing. Fi s , a e e alua ing which bio-ink had be -
e esul s, hey concluded ha he addi ion o HAMA conside ably imp o ed he p in -
abili y. Among he cells, he non-di e en ia ed ones showed highe ca ilage ECM p o-
duc ion, bu he e we e no signi ican di e ences in e ms o including hem in one ink o
he o he . Taking in o accoun hese da a, hey used GelMA/gellan gum/HAMA (GGH)
bio-ink o ab ica e sca olds wi h a middle/deep laye con aining MSCs and a supe icial
laye wi h ACPCs. As a esul , he laye ed sca old demons a ed posi i e s aining o
GAGs and collagen as well as chond ogenic gene exp ession (Figu e 10B). Howe e , he
mechanical p ope ies we e no speci ied, and he e we e no di e ences be ween he wo
di e en laye s; he e o e, u he s udies need o be conduc ed [72].
Figu e 10.
Laye ed sca olds: (
A
) Schema ic image o he manu ac u e o zonally s a i ied a icula
ca ilage. Adap ed om [
71
]. (
B
). His ological images o GAGs (sa anin-O,
op
), collagen ype II
(
middle
), and collagen ype I (
bo om
) ma ix o APCs and MSCs in GelMA/gellan gum/HAMA
(GGH) biop in ed sca olds a day 42. Scale ba = 100 µm. Adap ed om [72].
Pha maceu ics 2022,14, 1578 20 o 33
In ano he ecen wo k, Mouse e al. [
72
] aimed o de elop he e ocellula ca ilage
cons uc s by using h ee di e en cell ypes: equine chond ocy es, MSCs, and a icula
ca ilage p ogeni o cells (ACPC) which we e epo ed o be in he supe icial laye o he
ca ilage. To achie e his, cells we e embedded in o wo inks composed o GelMA/gellan
gum and GelMA/gellan gun/hyalu onic me hac yla e (HAMA). Then, he au ho s c ea ed
sca olds using ex usion-based biop in ing. Fi s , a e e alua ing which bio-ink had be e
esul s, hey concluded ha he addi ion o HAMA conside ably imp o ed he p in abili y.
Among he cells, he non-di e en ia ed ones showed highe ca ilage ECM p oduc ion,
bu he e we e no signi ican di e ences in e ms o including hem in one ink o he o he .
Taking in o accoun hese da a, hey used GelMA/gellan gum/HAMA (GGH) bio-ink o
ab ica e sca olds wi h a middle/deep laye con aining MSCs and a supe icial laye wi h
ACPCs. As a esul , he laye ed sca old demons a ed posi i e s aining o GAGs and
collagen as well as chond ogenic gene exp ession (Figu e 10B). Howe e , he mechanical
p ope ies we e no speci ied, and he e we e no di e ences be ween he wo di e en
laye s; he e o e, u he s udies need o be conduc ed [72].
3.2. 3D Biop in ing in Bone
OA is cha ac e ized by os eochond al damage ha a ec s ca ilage and bone issues.
Fo his eason, s udies ocused on he de elopmen o sca olds using 3D biop in ing ech-
nology o bone egene a ion ha e also been ca ied ou . The ideal sca old should ake in o
accoun he s uc u e and composi ion o human bone, and should no only ha e excellen
mechanical p ope ies, bu also con ain a po ous s uc u e, and be bo h os eoinduc i e and
os eoconduc i e. In addi ion, bone issue con ains a ascula sys em; he e o e, sca olds
should p o ide ascula iza ion o nou ish bone cells as well as ca ilage issue.
As a ha d issue, he mechanics and s i ness o he subs i u e sca old a e o key
impo ance. Consequen ly, many o he s udies use 3D p in ing echnology wi h syn he ic
ma e ials in which cells a e seeded la e on op o he ab ica ed sca old [
75
]. Among hem,
PCL has gained no o ie y due o i s good mechanical p ope ies, and because i a o s cell
adhesion and p oli e a ion [
76
]. In he ield o 3D biop in ing, he use o hyd ogels based
on polyme s such as algina e and GelMA is widesp ead, since hey a e good ca ie s o
he cells. Howe e , hey ha e shown low bioac i i y, and he biop in ed s uc u es a e
usually so and e y di e en om na i e bone’s mechanics. In o de o o e come hese
incon eniences, esea che s ha e been o ced o include o he elemen s, such as ce amics,
glasses, o ino ganic componen s (Table 3).
Pha maceu ics 2022,14, 1578 21 o 33
Table 3. Summa y o he 3D biop in ing s udies o bone egene a ion.
Bio-ink Cells Technique In Vi o Resul s Re .
ALG/PVA/HAP Mu ine cal a ia 3T3-E1 cells Ex usion-based biop in ing No
•PVA/HAP inc ease bio-ink heological
p ope ies
•Good cell iabili y a e p in ing
•Low mechanical p ope ies
[77]
Nano-HAP/ ype I COL Mu ine D1-MSCs Lase -based biop in ing Cal a ia de ec a s
•Manu ac u e o sca olds wi h wo geome ies:
ing and disk
•
High iabili y and p oli e a ion a e biop in ing
•Bone egene a ion in i o using disk sca olds
[78]
RGD-γ-i adia ed
ALG/nano-HAP pDNA
complexes encoding TGF-β3
and BMP-2 g ow h ac o s
Po cine BMSCs
Ex usion-based biop in ing +
PCL 3D p in ing Nude mice
•High cell iabili y using PCL co-p in ing
echnique
•High ans ec ion a es
•Bone ECM p oduc ion and mine aliza ion
•Bone o ma ion, imma u e os eoid de ec ion,
and ascula iza ion in i o
[79]
Vascula bio-ink:
RGD-γ-i adia ed
ALG/MC/nano-HAP
nanopa icles loaded wi h
VEGF
Os eoinduc i e bio-ink:
RGD-γ-i adia ed
ALG/MC/LAP/BMP-2
Po cine BMSCs
Ex usion-based biop in ing +
PCL 3D p in ing
Nude mice
and emo al-de ec a s
•Inc eased ascula iza ion in nude mice wi h
VEGF g adien sca olds
•Bone o ma ion and BMP-2 sus ained elease
wi h os eoinduc i e sca olds in nude mice
•Inc ease in essel olume and new bone
o ma ion using bo h bio-ink-based sca olds in
emo al-de ec a s
[80]
Type I COL/TCP
P eos eoblas cells
(MC3T3-E1)
And human ADMSCs
Ex usion-based biop in ing No
•Highly po ous sca olds
•Good cell iabili y and p oli e a ion a e
biop in ing
•TCP enhances sca old mine aliza ion a e
biop in ing wi h p eos eoblas cells
•TCP p omo es os eogenic ma ke s and gene
exp ession in hADMSCs a e biop in ing
[81]

Pha maceu ics 2022,14, 1578 22 o 33
Table 3. Con .
Bio-ink Cells Technique In Vi o Resul s Re .
ALG/GelMA/highly
angiogenic bo a e bioac i e
glass (13-93B3)
Human ADMSCs
Ex usion-based biop in ing +
PCL 3D p in ing No
•Glass enhances sca old s abili y a e
biop in ing by p omo ing algina e–GelMA
c osslinking
•
Glass solu es induces a pH inc ease in he media
ha is oxic o cells
[82]
ALG/GO Human MSCs Ex usion-based biop in ing No
•GO enhances bio-ink’s heological p ope ies
•P in abili y and sca old mechanics a e
imp o ed by GO
•GO p o ec s cells om oxida i e s ess and
p omo es hei di e en ia ion o bone
[83]
ALG/Gel/GO Human BMSCs Ex usion-based biop in ing No
•GO inc eases p in abili y and sca old ideli y
•
Good cell iabili y, p oli e a ion, and os eogenic
di e en ia ion a e biop in ing
•Highe GO concen a ions inc ease DNA
con en and mine aliza ion
[84]
Ac onyms—ALG: algina e; PVA: poly inyl alcohol; COL: collagen; HAP: hyd oxyapa i e; BMSCs: bone ma ow s em cells; PCL: polycap olac one; ECM: ex acellula ma ix; MC:
me hylcellulose; LAP: Laponi e; TCP: β- icalcium phospha e; ADMSCs: adipose-de i ed mesenchymal s em cells; GelMA: gela in me hac yla e; GO: g aphene oxide; Gel: gela in.
Pha maceu ics 2022,14, 1578 23 o 33
Hyd oxyapa i e (HAP), as a majo ino ganic componen o bone, has been ound
o be bioac i e and os eoinduc i e [
85
]. Fo his eason, i was included in he wo k
published by Bend sen e al. [
77
], who de eloped algina e/poly inyl alcohol (PVA)/HAP
inks o ob ain sca olds h ough ex usion-based biop in ing. The addi ion o PVA/HAP
enhanced he heological p ope ies as well as he iabili y o mu ine cal a ia 3T3-E1
cells. Impo an ly, al hough mechanical p ope ies inc eased wi h PVA/HAP, hey did no
esemble hose o bone [
77
]. Simila ly, Ke iquel e al. [
78
] e alua ed biop in ed sca olds
o bone egene a ion in a cal a ia de ec s. To do so, a nanohyd oxyapa i e/collagen
ype 1 bio-ink wi h mu ine D1-MSCs was de eloped, and sca olds wi h wo geome ies
( ing and disk) we e manu ac u ed using lase -based biop in ing. Resul s showed high cell
iabili y and p oli e a ion as well as bone egene a ion and o ma ion
in i o
, especially
when using disk geome y sca olds [
78
]. Despi e hese in e es ing esul s, biomechanics
we e no men ioned.
Fo os eochond al egene a ion, HAP has been also used. Fo example, Cunni e
e al. [
79
] ab ica ed an RGD-
γ
-i adia ed algina e and po cine bone ma ow MSC bio-ink.
In e es ingly, hey included nanohyd oxyapa i e complexed wi h plasmid DNA encoding
TGF-
β
3 and BMP-2 g ow h ac o s. Ex usion-based biop in ing was used accompanied
by PCL co-p in ing as a suppo ing mesh o p o ide mechanical s abili y o he cons uc
(Figu e 11)
. They achie ed good cell iabili y wi h his co-p in ing echnique, as well as
high ans ec ion a es. Mo eo e , highe ECM p oduc ion and mine aliza ion we e ob-
se ed wi h he plasmid-encoding g ow h ac o s. Finally, hey pe o med an
in i o
s udy
by implan ing he sca old subcu aneously in nude mice. As a esul , bone o ma ion, imma-
u e os eoid, and ascula iza ion we e de ec ed, sugges ing a easible app oach o bone e-
gene a ion [
79
]. In a ecen wo k by he same esea ch g oup, F eeman e al. [
80
] applied his
co-p in ing p ocedu e wi h bio-ink ex usion and PCL o manu ac u e sca olds. In his case,
wo di e en bio-inks we e p oposed: ascula and os eoinduc i e bio-inks. The ascula
bio-ink was composed o RGD-
γ
-i adia ed algina e/me hylcellulose and hyd oxyapa i e
nanopa icles loaded wi h he g ow h ac o VEGF. On he o he hand, he os eoinduc i e
bio-ink was based on RGD-
γ
-i adia ed algina e/me hylcellulose/Laponi e/BMP-2 and
po cine bone ma ow MSCs. The ob ained sca olds we e implan ed subcu aneously in
nude mice. VEFG g adien sca olds we e biop in ed wi h he ascula bio-ink, showing
ascula iza ion
in i o
. In con as , a e he implan a ion o he os eoinduc i e sca olds,
bone o ma ion and sus ained elease o BMP-2 due o Laponi e clay we e obse ed. In e -
es ingly, he sca olds con aining bo h bio-inks we e ab ica ed and e alua ed in a emo al
de ec s. Resul s showed an inc ease in essel olume as well as in new bone o ma ion,
indica ing a p omising he apeu ic app oach o bone egene a ion [
80
]. As a poin o
imp o emen , u he s udies on mechanical p ope ies should be pe o med.
Pha maceu ics 2022, 14, x FOR PEER REVIEW 24 o 34
Hyd oxyapa i e (HAP), as a majo ino ganic componen o bone, has been ound o
be bioac i e and os eoinduc i e [85]. Fo his eason, i was included in he wo k pub-
lished by Bend sen e al. [77], who de eloped algina e/poly inyl alcohol (PVA)/HAP inks
o ob ain sca olds h ough ex usion-based biop in ing. The addi ion o PVA/HAP en-
hanced he heological p ope ies as well as he iabili y o mu ine cal a ia 3T3-E1 cells.
Impo an ly, al hough mechanical p ope ies inc eased wi h PVA/HAP, hey did no e-
semble hose o bone [77]. Simila ly, Ke iquel e al. [78] e alua ed biop in ed sca olds o
bone egene a ion in a cal a ia de ec s. To do so, a nanohyd oxyapa i e/collagen ype 1
bio-ink wi h mu ine D1-MSCs was de eloped, and sca olds wi h wo geome ies ( ing
and disk) we e manu ac u ed using lase -based biop in ing. Resul s showed high cell i-
abili y and p oli e a ion as well as bone egene a ion and o ma ion in i o, especially
when using disk geome y sca olds [78]. Despi e hese in e es ing esul s, biomechanics
we e no men ioned.
Fo os eochond al egene a ion, HAP has been also used. Fo example, Cunni e e
al. [79] ab ica ed an RGD-γ-i adia ed algina e and po cine bone ma ow MSC bio-ink.
In e es ingly, hey included nanohyd oxyapa i e complexed wi h plasmid DNA encoding
TGF-β3 and BMP-2 g ow h ac o s. Ex usion-based biop in ing was used accompanied
by PCL co-p in ing as a suppo ing mesh o p o ide mechanical s abili y o he cons uc
(Figu e 11). They achie ed good cell iabili y wi h his co-p in ing echnique, as well as
high ans ec ion a es. Mo eo e , highe ECM p oduc ion and mine aliza ion we e ob-
se ed wi h he plasmid-encoding g ow h ac o s. Finally, hey pe o med an in i o
s udy by implan ing he sca old subcu aneously in nude mice. As a esul , bone o -
ma ion, imma u e os eoid, and ascula iza ion we e de ec ed, sugges ing a easible ap-
p oach o bone egene a ion [79]. In a ecen wo k by he same esea ch g oup, F eeman
e al. [80] applied his co-p in ing p ocedu e wi h bio-ink ex usion and PCL o manu ac-
u e sca olds. In his case, wo di e en bio-inks we e p oposed: ascula and os eoin-
duc i e bio-inks. The ascula bio-ink was composed o RGD-γ-i adia ed algi-
na e/me hylcellulose and hyd oxyapa i e nanopa icles loaded wi h he g ow h ac o
VEGF. On he o he hand, he os eoinduc i e bio-ink was based on RGD-γ-i adia ed al-
gina e/me hylcellulose/Laponi e/BMP-2 and po cine bone ma ow MSCs. The ob ained
sca olds we e implan ed subcu aneously in nude mice. VEFG g adien sca olds we e bi-
op in ed wi h he ascula bio-ink, showing ascula iza ion in i o. In con as , a e he
implan a ion o he os eoinduc i e sca olds, bone o ma ion and sus ained elease o
BMP-2 due o Laponi e clay we e obse ed. In e es ingly, he sca olds con aining bo h
bio-inks we e ab ica ed and e alua ed in a emo al de ec s. Resul s showed an inc ease
in essel olume as well as in new bone o ma ion, indica ing a p omising he apeu ic
app oach o bone egene a ion [80]. As a poin o imp o emen , u he s udies on me-
chanical p ope ies should be pe o med.
Figu e 11. Schema ic ep esen a ion o he biop in ing p ocess wi h co-p in ing o PCL and he bi-
op in ing o he bio-ink composed o algina e, MSCs, and nHAP-pDNA complexes. Adap ed om
[79].
Figu e 11.
Schema ic ep esen a ion o he biop in ing p ocess wi h co-p in ing o PCL and he
biop in ing o he bio-ink composed o algina e, MSCs, and nHAP-pDNA complexes. Adap ed
om [79].
Pha maceu ics 2022,14, 1578 24 o 33
Ano he bioce amic ha has been used o 3D biop in ing pu poses is
β
- icalcium
phospha e (TCP). Like HAP, TCP has been epo ed o p omo e os eogenic di e en ia ion
o MSCs; he e o e, Kim e al. [
81
] included his bioce amic in hei collagen- ype-I-based
bio-ink. Highly po ous sca olds we e ob ained h ough ex usion-based biop in ing.
Howe e , he mechanical p ope ies should ha e been imp o ed, since he na i e bone
alues we e no achie ed. Biological e alua ion was i s ca ied ou wi h p eos eoblas
cells (MC3T3-E1), showing good cell iabili y and p oli e a ion as well as enhancemen
o mine aliza ion a e biop in ing. Then, human adipose-de i ed MSCs we e used o
e alua e hei os eogenic di e en ia ion capaci y. As a esul , TCP-con aining sca olds
demons a ed ma ix mine aliza ion based on he inc ease in calcium and phospho us.
Fu he mo e, os eogenic ma ke s and os eogenic gene exp ession inc eased wi h TCP.
In e es ingly, os eogenic di e en ia ion was also shown in algina e/TCP/hMSCs sca olds
wi hou he need o adding an os eogenic cul u e medium [
81
]. Consequen ly, he use o
TCP could be a p omising app oach o he manu ac u e o sca olds o bone egene a ion.
Ne e heless, low mechanical p ope ies (5.94 MPa) make hese sca olds bes conside ed
as empo a y subs i u es o damaged bone.
Glass can also be inco po a ed in he ab ica ion o sca olds o bone egene a ion. In
he wo k p oposed by Kolan e al. [
82
], he au ho s included he highly angiogenic bo a e
bioac i e glass (13-93B3), which was app o ed by he FDA o he ea men o skin bu ns
and ch onic wounds. They de eloped a bio-ink composed o algina e/GelMA and human
adipose-de i ed MSCs using ex usion-based biop in ing o bio ab ica e he sca olds.
Like in he p e ious wo ks, hey ca ied ou a PCL co-p in ing p ocedu e o imp o e he
s abili y and mechanical p ope ies ( om 0.3 MPa o 50.6 MPa). Then, wo app oaches we e
p oposed: one consis ing o he addi ion o he glass o he PCL, and ano he consis ing o
he inclusion o he glass di ec ly in he bio-ink. Resul s showed a dec ease in cell iabili y
wi hin he days a e biop in ing when he glass was included wi hin he PCL. The au ho s
a gued ha he solubili y o he glass may ha e p oduced solu es ha inc eased he pH,
which was ha m ul o he cells. Fu he mo e, algina e/GelMA laye s los hei s abili y
o e ime, which accen ua ed he dec ease in cell iabili y. On he o he hand, when he
glass was added di ec ly o he bio-ink, an ini ial cell iabili y dec ease was obse ed due
o pH shock oxici y, bu cell eco e y was shown du ing he days a e biop in ing, since
he glass p omo ed c osslinking be ween he algina e and GelMA, making he sca olds
mo e s able. The au ho s concluded ha he glass could be in e es ing o manu ac u e
mo e s able sca olds, bu ha dynamic cul u e sys ems should be implemen ed o glass
concen a ion should be op imized in o de o a oid oxici y [82].
Ano he in e es ing app oach o bone egene a ion is he inco po a ion o ino ganic
componen s in o he bio-ink. Among hem, g aphene oxide (GO) has gained no o i-
e y because i s unc ional g oups enable he c ea ion o s ong in e ac ions wi h a ious
molecules [
83
,
86
]. Consequen ly, hyd ogels wi h high mechanical p ope ies ha e been
ob ained. Choe e al. [
83
] added GO in o a sodium algina e and human MSC bio-ink.
They showed ha by inc easing he GO concen a ion om 0.05 mg/mL o 1 mg/mL,
he bio-ink’s heological p ope ies, p in abili y (Figu e 12A), sca old s abili y, and me-
chanics inc eased. In e es ingly, GO p o ec s cells om oxida i e s ess, and p omo es
os eogenic di e en ia ion in e ms o alkaline phospha ase enzyme (ALP) p oduc ion and
mine aliza ion, along wi h os eogenic gene exp ession [
83
]. Likewise, Zhang e al. [
84
]
included di e en concen a ions o GO in hei bio-ink composed o sodium algina e,
gela in, and human bone ma ow MSCs. The GO imp o ed ex usion-based p in abili y
and sca old ideli y. Fu he mo e, as Figu e 12B shows, cell iabili y and p oli e a ion we e
good despi e inc easing GO concen a ions om 0.5 mg/mL o 2 mg/mL. Fu he mo e,
os eogenic di e en ia ion was shown in sca olds con aining GO. Impo an ly, highe GO
concen a ions imp o ed DNA con en as well as mine al olume a e bio eac o cul-
u e [
84
]. These wo s udies showed ha he use o GO could be an in e es ing op ion o
bone biop in ing, because i would no only imp o e he physical p ope ies o he sca old,
bu also p omo e os eogenesis.
Pha maceu ics 2022,14, 1578 25 o 33
Pha maceu ics 2022, 14, x FOR PEER REVIEW 26 o 34
Figu e 12. G aphene oxide sca olds: (A) Op ical images o he op iew o he p in ed sca olds,
indica ing be e p in abili y when GO inc eases om 0.05 mg/mL o 1 mg/mL. Scale ba s = 300 μm.
Adap ed om [83]. (B) Cell iabili y in he 3D-biop in ed GO sca olds a days 1, 7, and 42. Li ing
cells a e depic ed in g een, and dead cells a e in ed. Scale ba = 50 μm. Adap ed om [84].
3.3. 3D Biop in ing in Os eochond al Uni s
The la es and mos inno a i e app oach o egene a e os eochond al inju ies is he
manu ac u e o 3D s uc u es ha con ain bo h ca ilage and bone issues. Despi e he ac
ha his is a complica ed challenge due o all o he in insic cha ac e is ics ha each issue
mus mee , in e es ing and p omising ad ances ha e been achie ed (Table 4).
Figu e 12.
G aphene oxide sca olds: (
A
) Op ical images o he op iew o he p in ed sca olds,
indica ing be e p in abili y when GO inc eases om 0.05 mg/mL o 1 mg/mL. Scale ba s = 300
µ
m.
Adap ed om [
83
]. (
B
) Cell iabili y in he 3D-biop in ed GO sca olds a days 1, 7, and 42. Li ing
cells a e depic ed in g een, and dead cells a e in ed. Scale ba = 50 µm. Adap ed om [84].
3.3. 3D Biop in ing in Os eochond al Uni s
The la es and mos inno a i e app oach o egene a e os eochond al inju ies is he
manu ac u e o 3D s uc u es ha con ain bo h ca ilage and bone issues. Despi e he ac
ha his is a complica ed challenge due o all o he in insic cha ac e is ics ha each issue
mus mee , in e es ing and p omising ad ances ha e been achie ed (Table 4).
Pha maceu ics 2022,14, 1578 32 o 33
58.
Yang, X.; Lu, Z.; Wu, H.; Li, W.; Zheng, L.; Zhao, J. Collagen-Algina e as Bioink o Th ee-Dimensional (3D) Cell P in ing Based
Ca ilage Tissue Enginee ing. Ma e . Sci. Eng. C 2018,83, 195–201. [C ossRe ]
59.
Nguyen, D.; Hägg, D.A.; Fo sman, A.; Ekholm, J.; Nimking a ana, P.; B an sing, C.; Kaloge opoulos, T.; Zaunz, S.; Conca o, S.;
B i be g, M.; e al. Ca ilage Tissue Enginee ing by he 3D Biop in ing o iPS Cells in a Nanocellulose/Algina e Bioink. Sci. Rep.
2017,7, 658. [C ossRe ]
60.
Ra han, S.; Dejob, L.; Schipani, R.; Ha ne , B.; Möbius, M.E.; Kelly, D.J. Fibe Rein o ced Ca ilage ECM Func ionalized Bioinks
o Func ional Ca ilage Tissue Enginee ing. Ad . Heal hc. Ma e . 2019,8, 1801501. [C ossRe ]
61.
Vissche , D.O.; Lee, H.; an Zuijlen, P.P.M.; Helde , M.N.; A ala, A.; Yoo, J.J.; Lee, S.J. A Pho o-C osslinkable Ca ilage-De i ed
Ex acellula Ma ix Bioink o Au icula Ca ilage Tissue Enginee ing. Ac a Bioma e . 2021,121, 193–203. [C ossRe ]
62.
Zhang, X.; Liu, Y.; Luo, C.; Zhai, C.; Li, Z.; Zhang, Y.; Yuan, T.; Dong, S.; Zhang, J.; Fan, W. C osslinke -F ee Silk/Decellula ized
Ex acellula Ma ix Po ous Bioink o 3D Biop in ing-Based Ca ilage Tissue Enginee ing. Ma e . Sci. Eng. C.
2021
,118, 111388.
[C ossRe ]
63.
Li, Z.; Zhang, X.; Yuan, T.; Zhang, Y.; Luo, C.; Zhang, J.; Liu, Y.; Fan, W. Addi ion o Pla ele -Rich Plasma o Silk Fib oin Hyd ogel
Biop in ing o Ca ilage Regene a ion. Tissue Eng. Pa A 2020,26, 886–895. [C ossRe ]
64.
Singh, Y.P.; Bandyopadhyay, A.; Mandal, B.B. 3D Biop in ing using C oss-Linke -F ee Silk–Gela in Bioink o Ca ilage Tissue
Enginee ing. ACS Appl. Ma e . In e aces 2019,11, 33684–33696. [C ossRe ]
65.
Li, C.; Wang, K.; Zhou, X.; Li, T.; Xu, Y.; Qiang, L.; Peng, M.; Xu, Y.; Xie, L.; He, C.; e al. Con ollable Fab ica ion o Hyd oxybu yl
Chi osan/Oxidized Chond oi in Sul a e Hyd ogels by 3D Biop in ing Technique o Ca ilage Tissue Enginee ing. Biomed. Ma e .
2019,14, 025006. [C ossRe ]
66.
Gala aga, J.H.; Kwon, M.Y.; Bu dick, J.A. 3D Biop in ing Via an in Si u C osslinking Technique Towa ds Enginee ing Ca ilage
Tissue. Sci. Rep. 2019,9, 19987. [C ossRe ] [PubMed]
67.
Di Bella, C.; Duchi, S.; O’connell, C.D.; Blancha d, R.; Augus ine, C.; Yue, Z.; Thompson, F.; Richa ds, C.; Bei ne, S.; Ono illo,
C.; e al. In Si u Handheld Th ee-dimensional Biop in ing o Ca ilage Regene a ion. J. Tissue Eng. Regen. Med.
2017
,12, 611.
[C ossRe ] [PubMed]
68.
Ruiz-Can u, L.; Gleadall, A.; Fa is, C.; Segal, J.; Shakeshe , K.; Yang, J. Mul i-Ma e ial 3D Biop in ing o Po ous Cons uc s o
Ca ilage Regene a ion. Ma e . Sci. Eng. C. 2020,109, 110578. [C ossRe ] [PubMed]
69.
de Ruij e , M.; Ribei o, A.; Dok e , I.; Cas ilho, M.; Malda, J. Simul aneous Mic opa e ning o Fib ous Meshes and Bioinks o he
Fab ica ion o Li ing Tissue Cons uc s. Ad . Heal hc. Ma e . 2019,8, 1800418. [C ossRe ] [PubMed]
70.
Zhu, W.; Cui, H.; Boualam, B.; Masood, F.; Flynn, E.; Rao, R.D.; Zhang, Z.; Zhang, L.G. 3D Biop in ing Mesenchymal S em
Cell-Laden Cons uc wi h Co e–shell Nanosphe es o Ca ilage Tissue Enginee ing. Nano echnology
2018
,29, 158101. [C ossRe ]
71.
Wu, Y.; Ayan, B.; Moncal, K.K.; Kang, Y.; Dhawan, A.; Kodu u, S.V.; Ra nic, D.J.; Kamal, F.; Ozbola , I.T. Hyb id Biop in ing o
Zonally S a i ied Human A icula Ca ilage using Sca old-F ee Tissue S ands as Building Blocks. Ad . Heal hc. Ma e .
2020
,9,
2001657. [C ossRe ]
72.
Mouse , V.H.M.; Le a o, R.; Mensinga, A.; Dhe , W.J.A.; Gawli a, D.; Malda, J. Bio-Ink De elopmen o Th ee-Dimensional
Biop in ing o He e o-Cellula Ca ilage Cons uc s. J. Connec . Tissue Res. 2018,61, 137. [C ossRe ]
73.
La uen e-me chan, M.; Ruiz-alonso, S.; Zabala, A.; Gál ez-ma ín, P.; Ma chal, J.A.; Vázquez-lasa, B.; Gallego, I.; Saenz-del-bu go,
L.; Ped az, J.L. Chond oi in and De ma an Sul a e Bioinks o 3D Biop in ing and Ca ilage Regene a ion. Mac omol. Biosci.
2022
,
22, e2100435. [C ossRe ]
74.
O’connell, C.D.; Di Bella, C.; Thompson, F.; Augus ine, C.; Bei ne, S.; Co nock, R.; Richa ds, C.J.; Chung, J.; Gambhi , S.; Yue, Z.;
e al. De elopmen o he Biopen: A Handheld De ice o Su gical P in ing o Adipose S em Cells a a Chond al Wound Si e.
Bio ab ica ion 2016,8, 015019. [C ossRe ]
75.
Bha acha jee, A.; Bose, S. 3D P in ed Hyd oxyapa i e—Zn
2+
Func ionalized S a ch Composi e Bone G a s o O hopedic and
Den al Applica ions. Ma e . Des. 2022,221, 110903. [C ossRe ]
76.
Geno a, T.; Roa o, I.; Ca ossa, M.; Mo a, C.; Ca agne o, D.; Mussano, F. Ad ances on Bone Subs i u es h ough 3D Biop in ing.
In . J. Mol. Sci. 2020,21, 7012. [C ossRe ]
77.
Bend sen, S.T.; Quinnell, S.P.; Wei, M. De elopmen o a No el Algina e-poly inyl Alcohol-hyd oxyapa i e Hyd ogel o 3D
Biop in ing Bone Tissue Enginee ed Sca olds. J. Biomed. Ma e . Res. 2017,105, 1457. [C ossRe ]
78.
Ke iquel, V.; Oli ei a, H.; Rémy, M.; Ziane, S.; Delmond, S.; Rousseau, B.; Rey, S.; Ca os, S.; Amédée, J.; Guillemo , F.; e al. In Si u
P in ing o Mesenchymal S omal Cells, by Lase -Assis ed Biop in ing, o in Vi o Bone Regene a ion Applica ions. Sci. Rep.
2017,7, 1178. [C ossRe ]
79.
Cunni e, G.M.; Gonzalez-Fe nandez, T.; Daly, A.; Sa hy, B.N.; Jeon, O.; Alsbe g, E.; Kelly, D.J. Th ee-Dimensional Biop in ing o
Polycap olac one Rein o ced Gene Ac i a ed Bioinks o Bone Tissue Enginee ing. Tissue Eng. Pa A 2017,23, 891. [C ossRe ]
80.
F eeman, F.E.; Pi acco, P.; an Dommelen, L.H.A.; Nul y, J.; B owe, D.C.; Shin, J.Y.; Alsbe g, E.; Kelly, D.J. 3D Biop in ing
Spa io empo ally De ined Pa e ns o G ow h Fac o s o Tigh ly Con ol Tissue Regene a ion. Sci. Ad .
2020
,6, eabb5093.
[C ossRe ]
81.
Kim, W.; Kim, G. Collagen/Bioce amic-Based Composi e Bioink o Fab ica e a Po ous 3D hASCs-Laden S uc u e o Bone Tissue
Regene a ion. Bio ab ica ion 2019,12, 015007. [C ossRe ]
82.
Kolan, K.C.R.; Semon, J.A.; B ome , B.; Day, D.E.; Leu, M.C. Biop in ing wi h Human S em Cells-Laden Algina e-Gela in Bioink
and Bioac i e Glass o Tissue Enginee ing. In . J. Biop in . 2019,5, 3. [C ossRe ]

Pha maceu ics 2022,14, 1578 33 o 33
83.
Choe, G.; Oh, S.; Seok, J.M.; Pa k, S.A.; Lee, J.Y. G aphene Oxide/Algina e Composi es as No el Bioinks o Th ee-Dimensional
Mesenchymal S em Cell P in ing and Bone Regene a ion Applica ions. Nanoscale 2019,11, 23275–23285. [C ossRe ] [PubMed]
84.
Zhang, J.; Eyisoylu, H.; Qin, X.; Rube , M.; Mülle , R. 3D Biop in ing o G aphene Oxide-Inco po a ed Cell-Laden Bone
Mimicking Sca olds o P omo ing Sca old Fideli y, Os eogenic Di e en ia ion and Mine aliza ion. Ac a Bioma e .
2021
,121,
637–652. [C ossRe ] [PubMed]
85.
Kuma , A.; Ka goza , S.; Baino, F.; Han, S.S. Addi i e Manu ac u ing Me hods o P oducing Hyd oxyapa i e and Hyd oxyapa i e-
Based Composi e Sca olds: A Re iew. F on . Ma e . 2019,6, 313. [C ossRe ]
86.
Raslan, A.; Saenz del Bu go, L.; Ci iza, J.; Ped az, J.L. G aphene Oxide and Reduced G aphene Oxide-Based Sca olds in
Regene a i e Medicine. In . J. Pha m. 2020,580, 119226. [C ossRe ]
87.
Sun, Y.; You, Y.; Jiang, W.; Wang, B.; Wu, Q.; Dai, K. 3D Biop in ing Dual-Fac o Releasing and G adien -S uc u ed Cons uc s
Ready o Implan o Aniso opic Ca ilage Regene a ion. Sci. Ad . 2020,6, 37. [C ossRe ]
88.
Daly, A.C.; Kelly, D.J. Bio ab ica ion o Spa ially O ganised Tissues by Di ec ing he G ow h o Cellula Sphe oids wi hin 3D
P in ed Polyme ic Mic ochambe s. Bioma e ials 2019,197, 194–206. [C ossRe ]
89.
Bu dis, R.; Cha iye -P inz, F.; Kelly, D.J. Biop in ing o Biomime ic Sel -O ganised Ca ilage wi h a Suppo ing Join Fixa ion
De ice. Bio ab ica ion 2021,14, 015008. [C ossRe ]
90.
Deng, C.; Yang, J.; He, H.; Ma, Z.; Wang, W.; Zhang, Y.; Li, T.; He, C.; Wang, J. 3D Bio-P in ed Biphasic Sca olds wi h Dual
Modi ica ion o Silk Fib oin o he In eg a ed Repai o Os eochond al De ec s. Bioma e . Sci. 2021,9, 4891–4903. [C ossRe ]
91.
Kilian, D.; Ahl eld, T.; Akkineni, A.R.; Be nha d , A.; Gelinsky, M.; Lode, A. 3D Biop in ing o Os eochond al Tissue Subs i u es—
In Vi o-Chond ogenesis in Mul i-Laye ed Mine alized Cons uc s. Sci. Rep. 2020,10, 8277. [C ossRe ]
92.
Ki illo a, A.; Bushe , S.; Abubaki o , A.; Sukikh, G. Bioe hical and Legal Issues in 3D Biop in ing. In . J. Biop in .
2020
,6, 272.
[C ossRe ]
93.
Gilbe , F.; O’Connell, C.D.; Mladeno ska, T.; Dodds, S. P in Me an O gan? E hical and Regula o y Issues Eme ging om 3D
Biop in ing in Medicine. Sci. Eng. E hics 2017,24, 73–91. [C ossRe ]