Ma e ials Science & Enginee ing C 131 (2021) 112525
A ailable online 27 Oc obe 2021
0928-4931/© 2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
3D-p in ed algina e-hyd oxyapa i e ae ogel sca olds o bone
issue enginee ing
Ana Iglesias-Meju o, Ca los A. Ga cía-Gonz´
alez
*
Depa men o Pha macology, Pha macy and Pha maceu ical Technology, I+D Fa ma g oup (GI-1645), Facul y o Pha macy and Heal h Resea ch Ins i u e o San iago
de Compos ela (IDIS), Uni e sidade de San iago de Compos ela, E-15782 San iago de Compos ela, Spain
ARTICLE INFO
Keywo ds:
3D-p in ing
Supe c i ical d ying
Ae ogel
Bone sca old
Hyd oxyapa i e
ABSTRACT
3D-p in ing echnology allows he au oma ed and ep oducible manu ac u ing o unc ional s uc u es o issue
enginee ing wi h cus omized geome ies and composi ions by deposi ing ma e ials laye -by-laye wi h high
p ecision. Fo hese pu poses, he p oduc ion o bioac i e gel-based 3D-sca olds made o biocompa ible ma e-
ials wi h well-de ined in e nal s uc u e comp ising a dual (mesopo ous and mac opo ous) and highly in e -
connec ed po osi y is essen ial. In his wo k, ae ogel sca olds o bone egene a ion pu poses we e ob ained by
an inno a i e s a egy ha combines he 3D-p in ing o algina e-hyd oxyapa i e (HA) hyd ogels and he su-
pe c i ical CO
2
d ying o he gels. BET and SEM analyses we e pe o med o assess he ex u al pa ame e s o he
ob ained ae ogel sca olds and he dimensional accu acy o he o iginal compu e -aided design (CAD) design was
also e alua ed. The biological cha ac e iza ion o he ae ogel sca olds was also ca ied ou ega ding cell
iabili y, adhesion and mig a ion capaci y. The ob ained algina e-HA ae ogel sca olds we e highly po ous,
biocompa ible, wi h high ideli y o he CAD-pa e n and also allowed he a achmen and p oli e a ion o
mesenchymal s em cells (MSCs). An enhancemen o he ib oblas mig a ion owa d he damaged a ea was
obse ed in he p esence o he ae ogel o mula ions es ed, which is posi i e in e ms o bone egene a ion.
1. In oduc ion
Bone de ec s esul ing om umo s, in ec ions, auma, biochemical
diso de s and abno mal skele al de elopmen s gene a e se e e heal h
p oblems and ep esen high sociosani a y cos s o he na ional heal h
se ices [1]. Biological g a s (au og a s, allog a s) and non-
deg adable g a s (me allic and ce amic implan s) a e he mos com-
mon solu ions o hese pa hologies. Howe e , hei low a ailabili y ( o
biological g a s) and equen pos -su gical complica ions such as he
lack o osseoin eg a ion o he gene a ion o immune esponse ( o non-
deg adable g a s) a e among he impo an d awbacks ha may occu
[2]. Regene a i e medicine aims o ana omically and unc ionally
es o e damaged issues using a combina ion o (i) ad anced biode-
g adable 3D-sca olds as empo a y ex acellula ma ices (ECM), (ii)
cells, and (iii) g ow h ac o s [2,3]. The p ocessing me hod used o
ob ain hese bone sca olds should p o ide an op imum design o p o-
mo e he new issue o ma ion. These bone sca olds should ha e a well-
de ined in e nal s uc u e, dual in e connec ed po osi y and bioac i i y.
Gi en he inc easing complexi y in mo phology and composi ion o
hese s uc u es and he high sensi i i y o p ocessing en i onmen s (e.
g., high empe a u e, shea s ess, pH, use o o ganic sol en s), inno-
a i e and benign s a egies a e sough o gene a e hese new biomed-
ical p oduc s [4].
3D-p in ing is a cu ing-edge echnology ha allows he au oma ed
and ep oducible ab ica ion o unc ional, scalable and cus omized
a i icial s uc u es o pe sonalized medicine [5]. In gene al, his
echnology uses a laye -by-laye deposi ion o biocompa ible ma e ials
o achie e s able 3D-cons uc s. Speci ically, ex usion-based bio-
p in ing is an addi i e manu ac u ing echnique commonly used o
c ea e hyd ogel sca olds [6,7]. I is a p essu e-based me hod ha em-
ploys a obo ic sys em linked o a bioink dispensing sy inge. A
compu e -aided design (CAD) is used o deposi he bioink in a 3D-p e-
de ined shaped s uc u e. This echnique is able o p in di e en issues
wi h he gene a ion o ana omically co ec mac opo ous s uc u es [8].
A limi ed (bu g owing) numbe o bioma e ials a e sui able o bioink
o mula ions o gene a e 3D-p in ed sca olds as hey mus be p in able,
biocompa ible and p esen a ac i e s uc u al and mechanical p op-
e ies [9]. Ne e heless, s uc u es ob ained by 3D-p in ing ha e he
cus omized ex e nal mo phology essen ial o pe sonalized medicine,
bu hey usually lack he high p ecision equi ed o gene a e
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (C.A. Ga cía-Gonz´
alez).
Con en s lis s a ailable a ScienceDi ec
Ma e ials Science & Enginee ing C
jou nal homepage: www.else ie .com/loca e/msec
h ps://doi.o g/10.1016/j.msec.2021.112525
Recei ed 30 July 2021; Recei ed in e ised o m 18 Oc obe 2021; Accep ed 23 Oc obe 2021
Ma e ials Science & Enginee ing C 131 (2021) 112525
2
nanos uc u ed sca olds, wi h a con ol in he mac o and mic os uc-
u al le els. Fo his eason, he combina ion o 3D-p in ing wi h o he
echniques is needed o yield cus omized nanos uc u ed bioma e ials.
Polyme ic nanos uc u es wi h ad anced p ope ies can be ob ained
using supe c i ical luid echnology based on he use o supe c i ical
ca bon dioxide (scCO
2
) [10]. Namely, scCO
2
-assis ed d ying is adequa e
o he ex ac ion o he sol en o a gel while p ese ing he in e nal gel
s uc u al p ope ies, such as high po osi y and high speci ic su ace
a eas, in he so-called ae ogels. This echnology can be used o ob ain
ae ogel-con aining g a s and implan s [11], among hem ae ogel bone
sca olds o issue enginee ing applica ions [12–14]. Ae ogels p esen a
high mesopo osi y compa able o ha o he na i e ECM, which a o
cell g ow h, and can be ob ained om polysaccha ides and p o eins
[15].
Algina e is a na u al biopolyme widely used o bone sca olds and
o bioink o mula ion because i is biocompa ible, biodeg adable, non-
oxic and non-immunogenic, and suppo s cell g ow h [16]. Algina e
gene a es a s able hyd ogel in he p esence o low concen a ions o
di alen ca ions, namely Ca
2+
which p esen s he ad an age o i s low
oxici y wi h espec o o he di alen ions. Finally, algina e composi ion
esembles glycosaminoglycan (GAG) s uc u e, one o he majo com-
ponen s o he na u al ECM in human issue. Howe e , algina e-based
s uc u es usually lack bioac i i y and should be combined wi h o he
admix u es o bone issue enginee ing pu poses [17].
Hyd oxyapa i e (HA) is he majo ino ganic componen in mamma-
lian ha d issues and also an excellen bone subs i u e ma e ial because i
is bioac i e, os eoconduc i e, non- oxic and highly biocompa ible due o
i s chemical and s uc u al simila i y o na u al bone mine als [18,19].
This chemical simila i y is essen ial o apa i e deposi ion and subse-
quen bone egene a ion. HA may also ha e a ole as a c oss-linking
agen and as an ino ganic mechanical ein o ce [20]. Mo eo e , HA
and i s composi es a e sui able o a achmen , p oli e a ion and di -
e en ia ion o mesenchymal s em cells (MSCs). All hese p ope ies
ende HA a o able o os eoin eg a ion [21–23]. The sou ce and size o
HA pa icles is an impo an ac o on algina e-HA gel sca olds, because
he HA g ain size and speci ic su ace a ea a ec he gelling ime and
heological p ope ies o he hyd ogels. Mic oHA has been p oposed o
applica ions equi ing apid gela ion kine ics and imp o ed mechanical
p ope ies, while nanoHA is app op ia e o ende homogeneous
hyd ogels unde mo e con olled gela ion kine ics [20]. O e all,
algina e-HA composi es a e p omising candida es o bone issue engi-
nee ing whe e HA also igge s he mine aliza ion p ocess and s imu-
la es calcium phospha e and HA nanoc ys als nuclea ion and g ow h
[19,21].
S a egies o he gene a ion o a complex 3D-shaped s uc u es wi h
di e en unc ionali ies and a chi ec u es anging om he nanoscale o
he mac oscale a e unde de elopmen o gene a e s uc u es wi h his
po e hie a chy [24]. The design and op imiza ion o he inal po e
s uc u e (po e size hie a chy and p opo ions) is also essen ial o he
ma e ial pe o mance in issue enginee ing applica ions. The use o
ae ogel echnology can sol e some o he cu en 3D-p in ing limi a ions
ega ding he nanos uc u a ion o 3D-sca olds, bu he p oduc ion o
ae ogels wi h a cus omized ex e nal mo phology is a emaining
ema kable challenge [25]. Recen ly, se e al 3D-p in ed ae ogels ha e
shown an excellen p in abili y in he we s a e and he p ese a ion o
hei ex u al p ope ies like a high speci ic su ace a ea [25–27].
Ne e heless, ae ogels usually lack mac opo osi y and me hods o
con e i and o con ol he mac opo e size a e unde de elopmen
[28,53–55]. In gene al, ae ogels wi h a ious po e size anges a e highly
desi able o applica ions like bone issue enginee ing [29].
The unexplo ed combina ion o 3D-p in ing and ae ogel echnolo-
gies o bone issue enginee ing is he ein p oposed as a win-win s a egy
o ob ain bioma e ials wi h imp o ed pe o mance and a chi ec u e.
Finally, new s a egies o imp o e he ae ogels s eng hening and he
p in ing p ocess necessa y o ob ain mo e ine and in ica e s uc u es
a e s ill a challenge nowadays. In his wo k, 3D-p in ed algina e ae ogel
sca olds con aining hyd oxyapa i e we e p ocessed by a combina ion o
3D-p in ing o hyd ogels and supe c i ical CO
2
d ying o bone egen-
e a ion pu poses. The e ec s o algina e, HA and Ca
2+
concen a ions on
he 3D-p in ed hyd ogel sca olds p ocessabili y we e assessed. Then,
he 3D-p in ed ae ogels ob ained a e supe c i ical d ying we e e alu-
a ed ega ding hei ex u al (speci ic su ace a ea, po osi y, densi y,
sh inkage) and biological (cy o- and hemocompa ibili y, adhesion,
mig a ion) p ope ies. The ideli y o he di e en gel o mula ions wi h
espec o he o iginal CAD ile was also e alua ed.
2. Ma e ials and me hods
2.1. Ma e ials
Alginic acid sodium sal om b own algae wi h medium iscosi y
(gulu onic acid/mannu onic acid a io o 70/30, Mw 403 kDa, 3170
cps) and calcium chlo ide (CaCl
2
; M
w
110.98 g/mol, 99.99% pu i y)
we e p o ided by Sigma Ald ich (Mad id, Spain). Hyd oxyapa i e (HA;
M
w
502.31 g/mol, eagen g ade pu i y, mic opowde ) was p o ided by
Fluidino a (Mo ei a da Maia, Po ugal). CO
2
(pu i y >99.9%) was
supplied by Nippon Gases (Mad id, Spain) and absolu e e hanol (E OH)
by VWR (Radno , PA, USA). Wa e was pu i ied using e e se osmosis
( esis i i y >18 MΩ⋅cm; Milli-Q, Millipo e®, Mad id, Spain).
2.2. 3D-p in ing o hyd ogel sca olds
Aqueous bioinks wi h h ee di e en algina e concen a ions (6, 8
and 10 w % wi h espec o wa e ) we e p epa ed using milliQ wa e as a
sol en . Fu he mo e, di e en HA concen a ions (0, 8, 16 and 24 w %
wi h espec o wa e ) we e added o he 6 w % algina e solu ions. All
algina e solu ions we e p epa ed unde igo ous agi a ion (600 pm)
employing a homogenize (VWR os 60, Pennsyl ania, USA) o a leas
1 h a oom empe a u e (RT). The hus ob ained algina e solu ions we e
degassed in a sonica ion ba h (B anson 3510 Eme son, Fe guson, MO,
USA) o 10 min o elimina e ai bubbles. Hyd ogels we e ob ained by
p in ing he algina e inks wi h a Cellink BIOX Biop in e (Bos on, MA,
USA) a RT using an ex usion p in head wi h a 3-mL sy inge and a 410-
μ
m nozzle a he p in ing p essu e o 50 kPa and a p in ing eloci y o
12 mm/s. 3D-p in ed algina e hyd ogels wi h dimensions o 20x20x1
mm ( o physicochemical es s, c . Sec ions 2.4 and 2.5) and 10x10x1
mm ( o biological es s, c . Sec ions 2.6 o 2.8) we e ob ained using a
g id pa e n and 3 laye s. A e he p in ing p ocess, all sca olds we e
pu di ec ly in con ac wi h CaCl
2
aqueous solu ions (0.1, 0.5 and 1 M)
o gela ion (Fig. S1 o Supplemen a y In o).
2.3. Supe c i ical d ying o 3D-p in ed gels
Algina e hyd ogels u n alcogels by imme sion in absolu e E OH.
Two sol en exchanges o e hanol we e ca ied ou wi h an exchange
equency o 24 h. The alcogels we e w apped in il e pape and d ied
by supe c i ical d ying o ob ain he ae ogels. B ie ly, gels we e placed
in o a 100-mL s ainless s eel au ocla e (Tha P ocess, Pi sbu g, PA,
USA) and imme sed in 25 mL o absolu e e hanol o p e en hem om
sh inkage be o e being in con ac wi h comp essed CO
2
. scCO
2
was
supplied using a dual pis on pump and in oduced om he op o a
essel hea ed a he cons an empe a u e o 40 ◦C. Fi s ly, a con inuous
CO
2
low a e (5–7 g/min) a 120 ba ook place o 4 h. Ae ogels we e
hen ob ained a e CO
2
dep essu iza ion a a a e o 2 ba /min,
collec ed om he au ocla e and s o ed o cha ac e iza ion. The ob-
ained sca olds we e deno ed as Alg x%,HA y%,CaCl
2
zM (x =6, 8,
10 w %; y =8, 16, 24 w %; z =0.1, 0.5, 1 M).
2.4. Physicochemical cha ac e iza ion o algina e ae ogel sca olds
The skele al densi y o he ae ogels (
ρ
skel
) was de e mined using a
helium pycnome e (Quan ach ome, Boyn on Beach, FL, USA) a 25 ◦C
A. Iglesias-Meju o and C.A. Ga cía-Gonz´
alez
Ma e ials Science & Enginee ing C 131 (2021) 112525
3
and 1.01 ba . Values we e ob ained om i e eplica es. The appa en
densi y o he ae ogel sca olds o mula ions (
ρ
app
) was de e mined
ollowing he Eq. (1):
ρ
app =Ae ogel mass/Ae ogel olume (1)
whe e he olume o he ae ogels was ob ained by measu ing he h ee
dimensions o he ae ogel ob ained wi h a digi al calipe (Fowle ™,
New on, MA, USA). The po osi y o he s uc u es (
ε
) was ob ained om
Eq. (2) om he speci ic olume conside ing he ex e nal dimensions o
he ma e ial (V
) and he speci ic olume occupied by he oids in he
ull olume (V
oid,
).
ε
=(1−(
ρ
app/
ρ
skel)) x100 =(V oid, /V )x100 (2)
The inne po osi y o he ae ogel ibe s (
ε
) was ob ained om Eq. (3)
om he speci ic olume occupied by he ibe s (V
ibe s
) and he speci ic
olume occupied by he oids (i.e. po es) in he ibe s (V
oid, ibe s
).
ε
=(V oid, ibe s/V ibe s)x100 (3)
V oid, ibe s =V ibe s − (1/
ρ
skel)(4)
The olume ic sh inkage (in pe cen age) o he ae ogel sca olds
was calcula ed om he ex e nal dimensions o he ma e ial be o e and
a e supe c i ical d ying ollowing he Eq. (5):
Volume sh inkage = [(Alcogel olume–Ae ogel olume)/Alcogel olume ]x100
(5)
Low- empe a u e N
2
adso p ion/deso p ion analysis (ASAP 2000
Mic ome i ics Inc.; No c oss, GA, USA) was pe o med o assess ae ogel
ex u al p ope ies. Be o e measu emen s, samples we e degassed unde
acuum a 40 ◦C o 24 h. Speci ic su ace a ea (A
BET
) o ae ogels was
e alua ed employing he BET (B unaue –Emme –Telle ) me hod. Spe-
ci ic po e olume (V
p
), po e size dis ibu ions and mean po e diame e
(d
p
) we e de e mined applying he BJH (Ba e –Joyne –Halenda)
me hod. The mo phology o he ae ogels was e alua ed by scanning
elec on mic oscopy (SEM, EVO LS15, Zeiss, Obe kochen, Ge many).
Ae ogel samples we e i idium-spu e ed p io o imaging o minimize
cha ging and o imp o e he image quali y.
2.5. Dimensional accu acy es s
Two indices (p in ing accu acy and shape ideli y ac o –SFF–) we e
employed o assess he ideli y o he gel s uc u es ( o alcogels and
ae ogels) wi h espec o he o iginal p in ing ile p ocessed by 3D-p in -
ing in he i s s ep o he dual p ocessing s a egy he ein p oposed. The
p in ing accu acy and SFF we e calcula ed om Eqs. (6) and (7),
espec i ely:
P in ing accu acy (%) = [1− ((ǀAA–Aiǀ)/Ai ) ] x100 (6)
SFF =Ai/A(7)
whe e A
i
is he alcogel o ae ogel p in ed a ea espec i ely, and A is he
CAD a ea (p in ing ile).
2.6. Cell iabili y es s
The cy ocompa ibili y o he di e en algina e-HA ae ogel sca olds
was de e mined by assessing he iabili y o mouse emb yo ib oblas s
(BALB/c3 T3) a e 24 and 48 h o cul u e in he p esence o he ae ogel
o mula ions using he WST-1 es and in iplica e. This es is based on
he deg ada ion o WST-1 in o o mazan and is di ec ly co ela ed wi h
he numbe o me abolically ac i e cells. BALB cells (6500 cells/cm
2
)
we e seeded in 24-well pla es in DMEM supplemen ed wi h 15% e al
bo ine se um, penicillin 100 U/mL and s ep omycin 100 g/mL. Cells
we e incuba ed a 37 ◦C in a humidi ied a mosphe e en iched wi h 5%
CO
2
. Sca olds (1 ×1 ×0.1 cm) we e UV-s e ilized o 1 h and hen
placed in he wells wi h cells con aining 1500
μ
L o DMEM supplemen .
Posi i e con ols o cells wi h 1000
μ
L o medium and blanks o 1000
μ
L
o medium (bo h in iplica e) we e main ained a he same condi ions.
A e 24 and 48 h o cul u e, sca olds we e emo ed, 250
μ
L o medium
was le in he wells and 25
μ
L o WST 1 eagen was added. The pla e
was incuba ed o 2 h a he same condi ions and hen shaken ho -
oughly o 1 min. Finally, 110
μ
L we e ans e ed o a 96-well pla e o
measu e he abso bance a λ =450 nm in a mic opla e eade (In ini e®
M200, Tecan G oup L d., M¨
annedo , Swi ze land).
2.7. Cell a achmen es s
The sp eading o human bone ma ow mesenchymal s em cells
(MSCs) on o he sca olds was e alua ed by DAPI s aining o isualize
he cellula nuclei a e 6 and 13 days o cul u e [30]. MSC-seeded
sca olds we e ixed wi h pa a o maldehyde (4% w / ) o 10 min and
washed wi h PBS. Then, hey we e incuba ed in T i on/PBS (0.2% / )
solu ion o 5 min. Finally, sca olds we e washed wi h PBS, placed on
glass slides, and one d op o he DAPI-con aining P oLong gold an i ade
moun an (Molecula P obes Inc., Eugene, OR, USA) was added o each
sca old be o e hei s o age a −20 ◦C. Finally, cell a achmen in he
sca olds was e alua ed using a Leica TCS-SP2 spec al con ocal mic o-
scope (Leica TCS-SP2, Leica Mic osys ems Heidelbe g GmbH, Man-
nheim, Ge many) [31]. An open sou ce so wa e (ImageJ 1.5i, US
Na ional Ins i u es o Heal h, Be hesda, MD, USA) was used o analyze
he cap u ed images.
2.8. Mig a ion assay
Cell mig a ion was e alua ed by a sc a ch wound healing assay
[32,33]. A e he con luence, BALB cells monolaye was sc a ched by a
s aigh line using a s e ile pipe e ip and mimicking a wound in i o.
PBS was hen employed o wash cells and emo e cell deb is. The
di e en ae ogel sca old o mula ions we e placed in he wells wi h
esh cell cul u e medium a 37 ◦C. Pho og aphs o he sc a ch wound
we e ob ained wi h an in e ed mic oscope (Olympus, Japan) immedi-
a ely ( =0 h) and a 24 h o analyze he cell mig a ion. The mig a ion
a ea (%) was assessed as ollows (Eq. (8)):
Mig a ion a ea (%) = (A0–An)/A0×100 (8)
whe e A
0
ep esen s he ini ial wound a ea ( =0 h) and A
n
ep esen s
he esidual wound a ea a 24 h measu ed by ImageJ so wa e.
2.9. S a is ical analysis
Resul s o cell iabili y es s o each ae ogel sca old ype (n =3)
we e epo ed as mean alue ±s anda d de ia ion. - es s we e ca ied
ou o de e mine he s a is ical signi icance o he di e ences among he
g oups, and alues o p <0.05 we e conside ed as s a is ically
signi ican .
3. Resul s and discussion
3.1. E ec o algina e and CaCl
2
concen a ion on sca olds
The easible ope a ing egion o algina e ink o mula ion composi-
ion and Ca
2+
concen a ion anges o 3D-p in ing was i s ly de e -
mined. The ollowing c i e ia we e ollowed: (i) algina e bioink mus be
liquid-like in he p in head o a oid he ip clogging, bu (ii) i mus be
solid-like in he p in bed o main ain he cus omized 3D-geome y
du ing he deposi ion p ocess and a oid he s uc u al in eg i y loss.
Homogeneous 3D-s uc u es o med by di e en ia ed ilamen s a -
anged in laye s we e acco dingly ob ained wi h algina e concen a ions
in he 6–10 w % ange and a CaCl
2
concen a ion o 0.5 M (Fig. 1). Inks
A. Iglesias-Meju o and C.A. Ga cía-Gonz´
alez
Ma e ials Science & Enginee ing C 131 (2021) 112525
4
below o abo e his algina e concen a ion ange we e disca ded due o
he non-homogeneous appea ance ob ained o he 3D-p in ed hyd o-
gels. The 3D-s uc u e and po osi y, wi h mesopo es and mac opo es,
was clea ly ecognized in all ae ogels p oduced in he easible egion.
Tigh e ibe in e ac ions appea o be p esen a algina e concen a ions
o 10 w %. A highly in e connec ed s uc u e wi h mesopo es and
mac opo es was obse ed. An imp o emen in he ex u al pa ame e s
(A
BET
and V
p
) in all ae ogels was also obse ed (Table 1) wi h espec o
o he algina e ae ogels p e iously desc ibed [34].
Ionic c osslinking o algina e gels p o ided long- e m s abili y o
inal s uc u es in he 0.1–1 M CaCl
2
ange. Algina e sca olds c oss-
linked a his CaCl
2
concen a ion ange had a highly po ous s uc u e
h oughou he sca old wi h a high ae ogel su ace a ea (Fig. 2 and
Table 1). Finally, algina e sca olds c osslinked wi h CaCl
2
1 M solu ion
had mo e homogeneously p in ed s ands, which could be a ibu ed o
mo e Ca
+2
ions being in ol ed in he ionic c osslinking o he algina e
[35]. In gene al, ae ogel sca olds ob ained a algina e concen a ion
6 w % and geli ied wi h CaCl
2
1 M exhibi ed non-wo en ibe s wi h
igh e junc ions i compa ed o he es o o mula ions s udied and
esul ing in less b i le cons uc s.
High alues o ex u al p ope ies we e ob ained o all he es ed
ae ogel o mula ions, al hough he composi ion and c osslinke con-
cen a ion had an in luence in he esul s (Table 1), being he highes
alues ound in ae ogels ob ained om inks using an algina e concen-
a ion o 6 w % (Table 1). A simila end in e ms o ex u al p ope ies
was ob ained in ae ogels om algina e and algina e composi es, like
algina e-pec in mic osphe es gene a ed wi h di e en algina e- o-pec in
p opo ions [36].
3.2. E ec o hyd oxyapa i e concen a ion on sca old mo phology
Algina e hyd ogel p in ing esul ed in s able single ilamen s (Fig. 1),
bu wi hou signi ican binding be ween laye s. Mo eo e , algina e
ae ogel sca olds s a ed o dissol e jus a e hei imme sion in aqueous
solu ions losing hei in eg i y a e 24 h. The addi ion o HA o he
algina e ink was hypo hesized o p ese e he s ands geome y while
imp o ing s uc u e s abili y and bioac i i y o sca olds yielding an
imp o ed 3D-s uc u e o bone issue enginee ing. HA is hyd ophobic
and he sca olds would dissol e mo e g adually unde physiological
condi ions and main ain hei shape, a he expense o a educ ion in he
po osi y o he s uc u e [37,38]. To compensa e his, algina e ae ogels
we e he ein c osslinked a high concen a ions o he di alen ca ions
solu ion (CaCl
2
1 M), so ha mo e ca ions we e cap u ed inside he
sca olds and b idged he gel ne wo k. Finally, HA is in ol ed in p o ein
adso p ion and his p ocess causes cells anspo a ion in o he sca old,
which is posi i e because i may p omo e cell adhesion and p oli e a ion
on sca old su aces [39].
Homogeneous 3D-s uc u es we e ob ained o he HA concen a ion
ange es ed (0–24 w %) wi h ibe s and laye s consis en ly a anged.
The mac opo ous and mesopo ous s uc u es o he sca olds we e
main ained as well. A highe HA concen a ions, mo e HA g anules
appea o ming he ilamen s s acked oge he wi h he algina e (Fig. 3).
The easy handling o he syn hesized algina e-HA sca olds and he SEM
images showing binding be ween s ands appea o con i m he s uc-
u al in eg i y and s abili y o he ae ogels a e 3D-p in ing.
N
2
adso p ion-deso p ion analysis o algina e-HA ae ogel sca olds
show an impo an dec ease in he speci ic su ace a ea and o al po e
olume, while he po e diame e is almos unal e ed wi h espec o
sca olds ab ica ed wi h algina e only (Table 2). The dec ease in he
o al po e olume could be a ibu ed o he in e ac ions be ween –NH
3+
g oups o algina e and –OH g oups and Ca
2+
ions o HA ha lead o he
inal deposi ion o HA o e he sca old c ea ing an i egula su ace
s uc u e [16]. In gene al, he HA addi ion dec eased he ex u al
p ope ies in all algina e-HA o mula ions, due o he low mesopo osi y
o he HA powde (2 m
2
/g). Despi e ha , he ex u al p ope ies o he
HA-con aining algina e ae ogels may s ill be enough o a o cell seed-
ing and nu ien di usion h oughou he sca old s uc u e. The esul s
ob ained in e ms o ex u al pa ame e s a e he addi ion o HA a e
cohe en wi h p e ious s udies using o he polyme ic sca olds [16,40].
Appa en densi y alues inc eased g adually wi h he addi ion o HA
esul ing in a e e se e ec on po osi y, al hough all ae ogel sca old
o mula ions es ed eached alues abo e 80% (Table 2). The po osi y o
Fig. 1. SEM pic u es o 3D-p in ed algina e ae ogels p epa ed a CaCl
2
concen a ion o 0.5 M and om aqueous algina e inks o di e en concen a ions: a) 6, b) 8,
and c) 10 w %. Ae ogels a e obse ed a wo di e en magni ica ions.
Table 1
Tex u al p ope ies o ae ogel-based sca olds om inks a di e en algina e (6,
8, 10 w %) concen a ions and c osslinked in gela ion ba hs o di e en CaCl
2
(0.1, 0.5, 1 M) concen a ions. No a ion: A
BET
: speci ic BET su ace a ea, d
p
:
BJH-mean po e diame e , V
p
: BJH-speci ic po e olume.
Ae ogel sca old A
BET
(m
2
/g) d
p
(nm) V
p
(cm
3
/g)
Alg 6%, HA 0%, CaCl
2
0.1 M 438 ±22 22 ±1 3.14 ±0.16
Alg 6%, HA 0%, CaCl
2
0.5 M 277 ±14 22 ±1 2.00 ±0.10
Alg 6%, HA 0%, CaCl
2
1 M 183 ±9 19 ±1 1.16 ±0.06
Alg 8%, HA 0%, CaCl
2
0.1 M 180 ±9 17 ±1 0.95 ±0.05
Alg 8%, HA 0%, CaCl
2
0.5 M 177 ±9 22 ±1 1.00 ±0.05
Alg 8%, HA 0%, CaCl
2
1 M 147 ±7 21 ±1 0.97 ±0.05
Alg 10%, HA 0%, CaCl
2
0.1 M 172 ±9 18 ±1 1.04 ±0.05
Alg 10%, HA 0%, CaCl
2
0.5 M 158 ±8 16 ±1 0.87 ±0.04
Alg 10%, HA 0%, CaCl
2
1 M 219 ±11 14 ±1 1.11 ±0.06
A. Iglesias-Meju o and C.A. Ga cía-Gonz´
alez
Ma e ials Science & Enginee ing C 131 (2021) 112525
5
Fig. 2. SEM pic u es o 3D-p in ed algina e ae ogels p epa ed om 6 w % aqueous algina e inks and a di e en CaCl
2
concen a ions: a) 0.1, b) 0.5 and c) 1 M.
Ae ogels a e obse ed a wo di e en magni ica ions.
Fig. 3. Images and SEM pic u es o 3D-p in ed algina e-HA ae ogels p epa ed om 6 w % algina e inks wi h di e en HA concen a ions: (a) 0, (b, e) 8, (c, ) 16, and
(d, g) 24 w %. In all cases he gela ion was pe o med using 1 M CaCl
2
solu ions. Ae ogesl a e obse ed a wo di e en magni ica ions.
Table 2
Tex u al p ope ies o ae ogel-based sca olds p ocessed om 6 w % algina e inks wi h di e en HA concen a ions (0, 8, 16, 24 w %) and c osslinked wi h CaCl
2
1 M
solu ion. No a ion: A
BET
: speci ic BET su ace a ea, d
p
: BJH-mean po e diame e , V
p
: BJH-speci ic po e olume,
ρ
app
: appa en densi y,
ρ
skel
: skele al densi y,
ε
: sca old
po osi y,
ε
: ae ogel ibe s po osi y.
Ae ogel sca old A
BET
(m
2
/g)
d
p
(nm)
V
p
(cm
3
/g)
ρ
app
(g/ mL)
ρ
skel
(g/ cm
3
)
ε
(%)
ε
(%)
Alg 6%, HA 0%, CaCl
2
1 M 183 ±9 19 ±1 1.16 ±0.06 0.14 ±0.01 1.18 ±0.16 88.56 ±1.29 86.58 ±2.26
Alg 6%, HA 8%, CaCl
2
1 M 118 ±6 24 ±1 0.99 ±0.05 0.24 ±0.03 1.72 ±0.09 85.85 ±1.59 85.25 ±4.02
Alg 6%, HA 16%, CaCl
2
1 M 67 ±3 26 ±1 0.60 ±0.03 0.29 ±0.04 1.93 ±0.04 85.06 ±2.40 79.78 ±3.92
Alg 6%, HA 24%, CaCl
2
1 M 29 ±2 31 ±2 0.21 ±0.01 0.34 ±0.02 1.72 ±0.14 80.33 ±1.19 76.68 ±3.54
A. Iglesias-Meju o and C.A. Ga cía-Gonz´
alez

Ma e ials Science & Enginee ing C 131 (2021) 112525
6
he ibe s also dec eased wi h he addi ion o HA. The o al po osi y
alues o he sca olds (88.56 o 80.33%) a e sligh ly highe han he
ae ogel ibe s po osi y alues (86.58 o 76.68%) as he sca olds a e
o med by a 3D-mesh o e ically and ho izon ally aligned ae ogel
mic o ibe s sepa a ed by mac opo ous gaps. These a e good esul s since
he addi ion o HA ha e s ongly a ec ed o he ex u al pa ame e s
(A
BET
) bu no po osi y. Ne e heless, highe po osi y (80–99%) and
lowe densi ies (0.02 g/ cm
3
as bulk densi y) alues we e p e iously
epo ed o algina e ae ogel in he o m o mic opa icles [41–43].
Consis en ly, he inco po a ion o HA has ad an ages (bioac i i y, sim-
ila i y o human bone mine al phase) [18,19] a he expense o a sligh
dec ease in ex u al pa ame e s.
The olume ic sh inkage o ae ogel sca olds a e pe o ming he
supe c i ical d ying was es ed (Fig. 4) o un eil he in luence o his
p ocess in he end ae ogel sca olds. The di ec sol en exchange o
e hanol esul ed in ae ogels wi hou high olume ic sh inkage alues
and wi h educed o e all p ocessing ime. S uc u es con aining HA had
a lowe olume sh inkage wi h espec o he o mula ion wi hou HA
(Alg 6%, HA 0%, CaCl
2
1 M). These a e good esul s since highe olume
sh inkage alues (42–65%) ha e been epo ed o algina e ae ogels
ob ained by sequen ial sol en exchanges [44].
3.3. Dimensional accu acy es s
P in ing accu acy and SFF indices we e employed o assess he
ep oducibili y and he ideli y o he dual p ocessing s a egy he ein
p oposed. The alues ob ained o bo h pa ame e s a e highe when
assessed in he alcogels han when assessed in he ae ogels (Table 3).
This ea u e was expec ed since he olume sh inkage epo ed in Sec-
ion 3.2 in luences he dimensional accu acy indices. None heless, he
alcogel p in ing accu acy and SFF alues ob ained a e close o he alues
p e iously epo ed o 3D-p in ed algina e gels and o o he poly-
saccha ide gels [37,45].
3.4. Biocompa ibili y es s o ae ogel sca olds
BALB cells we e cul u ed in he p esence o algina e-HA ae ogel
sca olds o cell cy o oxici y e alua ion. Fig. 5 ep esen s he cell
iabili y a e cul u e o 24 and 48 h wi h alues eaching ca. 100% o
algina e ae ogels, which means ha hey had no nega i e e ec on cell
p oli e a ion. Simila esul s on BALB cells iabili y a e 48 h we e
ound o algina e-HA ae ogels, ega dless o he HA concen a ion.
In gene al, hese iabili y esul s indica e ha nei he he sca old
composi ion no he dual p ocessing s a egy comp omised he cell
iabili y. The high cell iabili y obse ed a e he es s con i med he
non- oxici y o he ae ogel s uc u es and co ela es wi h sca olds
con aining he same componen s [46]. Indeed, he biocompa ibili y o
he ini ial componen s o he sca olds (algina e and HA) was al eady
epo ed [47]. O e all, hese a e p omising esul s since cy ocompa i-
bili y and a sui able mic os uc u e (po e size and po osi y) go e n he
p ope ies o an ideal sca old.
3.5. Cell a achmen and p oli e a ion es s on ae ogel sca olds
A challenge in issue enginee ing is he gene a ion o bioma e ials
wi h an adequa e s uc u e o ac as a subs a e o cell bonding,
mul iplica ion, de elopmen and ep oduc ion [48]. MSCs a achmen
and p oli e a ion in ae ogels we e he ein es ed as his cell line is
a ac i e o issue enginee ing applica ions aiming a he gene a ion o
a i icial bone subs i u es. MSCs p esen sel - enewal po en ial, hey can
di e en ia e in i o and ex i o in o di e en cell ypes like chond ocy es
o os eoblas s and hei isola ion is no ela ed o dono -si e mo bidi y
[49]. In he case o os eoblas s, hei a achmen o sca olds depends on
0
10
20
30
40
50
60
Alg 6%,
HA 0%,
CaCl₂ 1M
Alg 6%,
HA 8%,
CaCl₂ 1M
Alg 6%,
HA 16%,
CaCl₂ 1M
Alg 6%,
HA 24%,
CaCl₂ 1M
Volume sh inkage (%)
Fig. 4. Volume sh inkage (in pe cen age) o di e en algina e-HA
o mula ions.
Table 3
P in ing ideli y indices o ae ogels p epa ed om algina e-HA o mula ions.
Ae ogels we e manu ac u ed a an algina e concen a ion o 6 w %, a di e en
HA concen a ions (0, 8, 16 and 24 w %) and a a CaCl
2
concen a ion o 1 M.
Ae ogel
sca old
Alcogel P in ing
Accu acy (%)
Ae ogel P in ing
Accu acy (%)
Alcogel SFF Ae ogel SFF
Alg 6%,
HA 0%,
CaCl₂
1 M
77.73 ±11.03 43.56 ±12.94 0.82 ±0.08 0.64 ±0.05
Alg 6%,
HA 8%,
CaCl₂
1 M
75.51 ±8.54 39.06 ±10.70 0.81 ±0.05 0.62 ±0.04
Alg 6%,
HA 16%,
CaCl₂
1 M
83.51 ±2.29 50.18 ±6.05 0.86 ±0.02 0.67 ±0.03
Alg 6%,
HA 24%,
CaCl₂
1 M
83.35 ±6.21 48.14 ±4.84 0.86 ±0.05 0.66 ±0.02
0
20
40
60
80
100
120
140
160
Posi e
Con ol
Alg 6%,
HA 0%,
CaCl₂ 1M
Alg 6%,
HA 8%,
CaCl₂ 1M
Alg 6%,
HA 16%,
CaCl₂ 1M
Alg 6%,
HA 24%,
CaCl₂ 1M
Cell Viabili y (%)
Fig. 5. Cell iabili y es s o algina e-HA ae ogels. Viabili y (exp essed in %) o
BALB cells a e 24 (g ey ba s) and 48 h (black) o con ac wi h sca olds
de e mined by he WST-1 es . The e was no s a is ically signi ican di e ences
among g oups ( - es ; p <0.05).
A. Iglesias-Meju o and C.A. Ga cía-Gonz´
alez
Ma e ials Science & Enginee ing C 131 (2021) 112525
7
he su ace a ea and he po osi y o he ma e ials.
No di e ences in MSCs a achmen and sp eading we e obse ed
be ween ae ogel o mula ions in e ms o nuclei s aining (Fig. 6). The
p esence o HA in he sca olds did no in luence MSCs a achmen as
p e iously obse ed o s uc u es wi h simila composi ion [46,49].
Ne e heless, o highe HA concen a ions (16 and 24 w %) he
a achmen dec eases a 6 and 13 days p obably due o he high su-
pe icial oughness and i egula i y ha gene ally a o cell a achmen
(HA concen a ion o 8 w %) bu a such highe le els i could make i
ha d o MSCs o p oli e a e ully a ached o he ae ogel.
A p oli e a ion endency is obse ed o all ae ogel o mula ions,
since mo e nuclei we e s ained a e cul u ing sca olds o 13 days han
a e 6 days (Fig. 6). This ea u e indica es ha sca olds we e non- oxic,
biocompa ible, sui able o he a achmen and g ow h o MSCs and able
o p omo e cell adhesion and p oli e a ion, as well as he cell coloni-
za ion o he sca old. Small po e diame e s and high su ace a eas as
ob ained o he ae ogels o his wo k a e sui able o os eoblas
a achmen because hese condi ions allow cell in e pene a ion.
3.6. Cell mig a ion es
Fig. 7a– shows he e olu ion o he wound closu e p ocess a e he
sc a ch es wi h BALB cells in he p esence o di e en ae ogel o mu-
la ions. Fig. 7g shows he inc ease in BALB cell mig a ion in he p esence
o ae ogel sca olds wi h espec o he posi i e con ol. These a e good
esul s since he well-known bioine p ope ies o algina e make i ha d
o be deg aded in i o and his ea u e could ha e es ic ed cellula
adhesion and mig a ion as p e iously epo ed [50]. In gene al, s uc-
u es ha a e s able, wi h sa is ac o y mo phology and po e size allow
signi ican human ib oblas mig a ion in wound closu e in i o assays
[51]. Ne e heless, inding he bes mic oa chi ec u e o cell mig a ion
in issue egene a ion is s ill challenging. In p e ious s udies, human
de mal ib oblas mig a ion wi h algina e composi es showed a high
closu e o he sc a ch [51]. Fu he mo e, algina e composi es combined
wi h Ca
2+
ca ions esul ed in an enhancemen on ib oblas s mig a ion
like he he ein obse ed, p obably due o changes in he egula ion o
genes in ol ed in he wound healing p ocess ca ied ou by ib oblas s.
Finally, as chemo axis is an inhe en esponse o cells i was hypo he-
sized o be in ol ed in he cell mig a ion p ocess since he highe con-
cen a ion o biochemical ac o s sec e ed by inju ed issues ac i a e he
su ounding cells and s imula e he mig a ion o cells [52]. This bio-
logical esponse could also play a signi ican ole in he inc ease o he
mig a ion o ib oblas s in he p esence o algina e-based ae ogels he ein
obse ed.
4. Conclusions
Algina e-HA ae ogel sca olds we e success ully ab ica ed wi h a
p ecise and cus omized nanos uc u e by he combina ion o 3D-p in -
ing and ae ogel echnologies. This echnological combina ion esul ed
Fig. 6. Con ocal mic oscopy images o DAPI-s ained MSCs seeded on ae ogel sca olds and cul u ed o (a) 6, and (b) 13 days. (c) Cell a achmen and p oli e a ion
es s o algina e-HA ae ogels a e 6 (g ey ba s) and 13 (black) days o cul u e in MSCs.
A. Iglesias-Meju o and C.A. Ga cía-Gonz´
alez
Ma e ials Science & Enginee ing C 131 (2021) 112525
8
in nanos uc u ed and dual po ous algina e ae ogels o in e es o issue
enginee ing applica ions. 3D-s uc u e is conse ed a e all p ocessing
s eps and algina e, HA and CaCl
2
concen a ions de e mine sca old
ex u e. An op imized sca old o mula ion mus p o ide a long- e m
s abili y and he equi ed biocompa ibili y o issue enginee ing ap-
plica ions. Cell iabili y es s e ealed no oxici y e ec o nega i e
impac on BALB cells on no mal cell en i onmen because o he p es-
ence o he sca old o mula ions. Fu he mo e, ib oblas s also suc-
cess ully accomplished he mig a ion p ocess when cul u ed wi h he
ae ogel o mula ions he ein p oposed. MSCs we e able o a ach and
p oli e a e on algina e-HA ae ogels upon cul u e o 6 and 13 days. All
hese ea u es a e highly desi able o sca olds o ac as empo a y
suppo s and ha bo he bone egene a ion p ocess. Finally, he ideli y
o he end ae ogel sca olds wi h espec o he o iginal CAD is high.
Consis en ly, he mac opo ous algina e-HA ae ogel sca olds ob ained
by his no el echnological combina ion o 3D-p in ing and supe c i ical
d ying me hods ep esen a p omising al e na i e o bone egene a i e
applica ions opening up new possibili ies o pe sonalized medicine.
O he equi emen s o accomplish bone issue o ma ion like he
deg ada ion a e o he 3D-s uc u es and hei mechanical p ope ies
will be op imized in a u u e wo k.
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.msec.2021.112525.
Acknowledgmen s
This wo k was suppo ed by Xun a de Galicia [ED431C 2020/17],
MICINN [PID2020-120010RB-I00], Agencia Es a al de In es igaci´
on
[AEI] and FEDER unds. Wo k ca ied ou in he amewo k o he COST
Ac ion CA18125 “Ad anced Enginee ing and Resea ch o ae oGels o
En i onmen and Li e Sciences” (AERoGELS) and unded by he Eu o-
pean Commission. A.I.-M. acknowledges o Xun a de Galicia o he
p edoc o al esea ch ellowship [ED481A- 2020/104]. A. Alonso is
commended o his echnical con ibu ion o his wo k.
Decla a ion o compe ing in e es
Au ho s decla e no con lic o in e es .
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