Enhancing alginate dialdehyde-gelatin (ADA-GEL) based hydrogels for biofabrication by addition of phytotherapeutics and mesoporous bioactive glass nanoparticles (MBGNs)

Bioma e ials o D ug Deli e y
Jou nal o Bioma e ials Applica ions
2025, Vol. 39(6) 524–556
© The Au ho (s) 2024
A icle euse guidelines:
sagepub.com/jou nals-pe missions
DOI: 10.1177/08853282241280768
jou nals.sagepub.com/home/jba
Enhancing algina e dialdehyde-gela in
(ADA-GEL) based hyd ogels o
bio ab ica ion by addi ion o
phy o he apeu ics and mesopo ous
bioac i e glass nanopa icles (MBGNs)
Faina Bide
1
, Chia a Gunnella
1,2,
*, Jana T Reh
1,
*, Co ina-Elena Clejanu
1
, Sonja Ku h
1
,
Ana M Bel ´
an
3
and Aldo R Boccaccini
1

Abs ac
This s udy explo es he 3D p in ing o algina e dialdehyde-gela in (ADA-GEL) inks inco po a ing phy o he apeu ic agen s,
such as e ulic acid (FA), and silica e mesopo ous bioac i e glass nanopa icles (MBGNs) a wo di e en concen a ions.
3D sca olds wi h bioac i e p ope ies sui able o bone issue enginee ing (TE) we e ob ained. The deg ada ion and
swelling beha iou o films and 3D p in ed sca olds indica ed an accele a ed end wi h inc easing MBGN con en , while FA
appea ed o s abilize he samples. De e mina ion o he deg ee o c osslinking alida ed he inc eased s abili y o hyd ogels
due o he addi ion o FA and 0.1% (w/ ) MBGNs. The inco po a ion o MBGNs no only imp o ed he e ec i e moduli and
con e ed bioac i e p ope ies h ough he o ma ion o hyd oxyapa i e (HAp) on he su ace o ADA-GEL-based samples
bu also enhanced VEGF-A exp ession o MC3T3-E1 cells. The beneficial impac o FA and low concen a ions o MBGNs in
ADA-GEL-based inks o 3D (bio)p in ing applica ions was co obo a ed h ough a ious p in ing expe imen s, esul ing in
highe p in ing esolu ion, as also confi med by heological measu emen s. Cy ocompa ibili y in es iga ions e ealed
enhanced MC3T3-E1 cell ac i i y and iabili y. Fu he mo e, he p esence o mine al phases, as confi med by an in i o
biomine aliza ion assay, and inc eased ALP ac i i y a e 21 days, a ibu ed o he addi ion o FA and MBGNs, we e
demons a ed. Conside ing he acqui ed s uc u al and biological p ope ies, along wi h e ficien d ug deli e y capabili y,
enhanced biological ac i i y, and imp o ed 3D p in abili y, he newly de eloped inks exhibi p omising po en ial o
bio ab ica ion and bone TE.
Keywo ds
Hyd ogels, bioac i e glass pa icles, phy o he apeu ic agen , 3D (bio)p in ing, d ug deli e y, bone issue enginee ing
In oduc ion
The goal o issue enginee ing (TE) is he egene a ion o
new issues by he sma combina ion o cells, bioma e ials
and g ow h ac o s.
1
Especially, he field o bone TE is
cu en ly in an exci ing phase ma ked by significan
esea ch e o s di ec ed owa ds he de elopmen o in-
no a i e and imp o ed egene a i e bioma e ials.
2
To ad-
d ess he demand o inno a i e medical echnology and
al e na i e app oaches in TE, h ee-dimensional (3D)
sca olds con inue o be a he cen e o esea ch. One
me hod cu en ly employed o p oducing 3D sca olds is
3D biop in ing.
3
This me hod in ol es an addi i e
manu ac u ing p ocess ha equi es he use o a 3D bio-
p in e and sui able bioma e ials (bioinks) o cons uc 3D
1
Ins i u e o Bioma e ials, F ied ich-Alexande Uni e si y E langen-
Nu embe g, E langen, Ge many
2
Depa men o Elec onics, In o ma ion and Bioenginee ing, Poli ecnico di
Milano, Milano, I aly
3
Depa amen o de Ingenie ´
ıa y Ciencia de los Ma e iales y del T anspo e.
Escuela Poli ´
ecnica Supe io , Vi gen de ´
A ica 7, Uni e sidad de Se illa,
Se ille (Spain)
*These wo au ho s con ibu ed equally o his wo k.
Co esponding au ho :
Aldo R Boccaccini, Ins i u e o Bioma e ials, F ied ich-Alexande
Uni e si y E langen-Nu embe g, Caue s asse 6, 91058 E langen,
Ge many.
Email: [email p o ec ed]
sca olds h ough a laye -by-laye ex usion p ocess.
4
Bi-
oinks ypically consis o hyd ogels de i ed om bio-
compa ible polyme s, chosen o hei no able a ibu es
such as app op ia e mechanical p ope ies, biodeg adabili y,
and p in abili y. Addi ionally, bioinks mus es ablish a
sui able en i onmen o cell p oli e a ion and a achmen ,
acili a ing he de elopmen o 3D cons uc s ha mimic
human na i e issue.
5
Ne e heless, achie ing an op imal
balance be ween sui able p in ing cha ac e is ics, mechan-
ical s abili y, deg adabili y, and biocompa ibili y o bioinks
is s ill challenging. Chi osan, collagen, gela in and algina e
a e na u al polyme s equen ly used o de elop bioinks o
3D biop in ing. These ma e ials exhibi no ewo hy p op-
e ies ha make hem sui able o eplica ing he cha ac-
e is ics o he na i e ex acellula ma ix (ECM).
6
Algina e,
a na u al polyme ex ac ed om b own algae, is employed
in d ug deli e y s a egies and 3D p in ing app oaches.
7,8
The algina e polyme chain consis s o polysaccha ide,
o ming a linea s uc u e wi h (1,4)-linked β-D-mannu onic
(M) and α-L-gulu onic acid (G) esidues. The G blocks ha e
he po en ial o unde go ionic c osslinking in he p esence o
di alen ca ions such as s on ium (S
2+
)o Ca
2+
, esul ing
in he o ma ion o hyd ogels.
7
Fu he mo e, algina e is
ecognized o i s widesp ead a ailabili y, excellen bio-
compa ibili y, and ela i ely low cos .
8,9
Howe e , he slow
deg ada ion a e o algina e hyd ogels poses a limi a ion.
This challenge has been add essed by u ilizing he oxidized
o m o algina e known as algina e dialdehyde (ADA).
10
As
a esul o he oxida ion p ocess, he e is a educ ion in
molecula weigh , leading o an accele a ed deg ada ion a e
compa ed o unmodified algina e. Addi ionally, he oxi-
da ion p ocess gene a es ee aldehyde g oups, allowing
hem o o m bonds wi h amino (NH
2
)-g oups in p o ein
molecules, such as gela in (GEL), h ough Schi ’s base
o ma ion.
8,11
GEL is bo h bio eso bable and biocompa i-
ble, con aining a ginine–glycine–aspa ic acid (RGD) se-
quences. The p esence o RGD sequences in gela in gi es i
he abili y o enhance cell a achmen and acili a e cell-cell
in e ac ion.
12
Mic obial ansglu aminase (mTG) is an en-
zyme na u ally p esen in he human body, playing a ole in
aiding blood coagula ion and s abilizing p o ein
s uc u es.
13,14
mTG assis s in he c osslinking o s uc u al
p o eins by c ea ing bonds be ween he NH
2
-g oups o
glu amine and lysine in collagen, esul ing in a sui able
c osslinke o GEL.
13,15
In ecen imes, he e has been a g owing in e es in
in eg a ing he bal d ugs in o enginee ed bioma e ials, due o
he acknowledged biological unc ionali ies o hese phy-
o he apeu ic agen s.
16
Specifically, he inclusion o com-
pounds wi h phenolic ings, such as e ulic acid (FA),
eme ges as a a o able and p omising s a egy.
17
FA (4-
hyd oxy-3-me hoxycinnamic acid) is a na u ally occu ing
compound ound in plan s, p esen in lea es, seeds, and
plan cell walls. I has he capabili y o o m co alen
c osslinks wi h p o ein g oups, such as lysine, ha a e
p esen in GEL.
18,19
Mo eo e , FA is widely ecognized o
i s an iaging, an icance , an idiabe ic, an ioxida i e, and
an ibac e ial cha ac e is ics.
17
In ou p e ious s udy
20
he
an ibac e ial e ec o FA inco po a ed in ADA-GEL hy-
d ogels was al eady p o en using S. epide midis s ains. A
educ ion o colony o ming uni s (CFU) on aga pla es due
o he addi ion o FA was ound. A u bidi y es wi h S.
au eus as G am-posi i e bac e ia and E. coli as G am-
nega i e bac e ia has shown he an ibac e ial e ec o FA
wi hin ADA-GEL hyd ogels. Mo eo e , FA has demon-
s a ed po en ial in TE, especially o applica ions like skin
egene a ion o in skinca e o mula ions. On he o he hand,
he explo a ion o mul i unc ional sca olds inco po a ing
FA in hyd ogels, pa icula ly o bone TE, has no been
ex ensi ely in es iga ed. To he bes o ou knowledge, only
one publica ion by Anjali e al.
21
has epo ed he in e-
g a ion o FA in o a sel -c osslinking ADA-GEL sys em,
u ilizing i as an an imic obial compound o exuda ing
wounds.
21
In his wo k FA was inco po a ed in o ADA-
GEL-based hyd ogels, emphasizing he significance o
phy o he apeu ic agen s wi hin hyd ogels o biomedical
applica ions and expanding he unde s anding o such
composi es. Fu he mo e, his wo k aimed o add ess spe-
cific challenges associa ed wi h na u al hyd ogels, such as
mechanical weakness and limi ed bioac i e p ope ies. To
o e come his limi a ions, we inco po a ed ino ganic bio-
ac i e nanopa icles in o ADA-GEL-based-hyd ogels.
These nanopa icles no only ha e he po en ial o enhance
he mechanical p ope ies o hyd ogels bu also may
s imula e he nuclea ion and subsequen g ow h o apa i e
c ys als on he su ace o sca olds, mimicking bone.
22–24
A
p omising class o ino ganic pa icles a e mesopo ous
bioac i e glass nanopa icles (MBGNs) in he SiO
2
-CaO
sys em, which a e p oduced using sol-gel app oaches.
25,26
Gene ally, bioac i e nanopa icles find applica ions in ha d
TE, as ca ie s o d ug deli e y, o as coa ings o implan s,
pa icula ly due o hei biocompa ibili y and os eos imu-
la ing p ope ies.
25,27,28
In his wo k, MBGNs (nominal
composi ion: 70% SiO
2
, 30% CaO, mol%) we e u ilized,
which a e known o hei significan ly la ge po e olume
and specific su ace a ea. Addi ionally, hese pa icles can
be syn hesized in cus omized sizes and shapes and hey can
be loaded wi h a sui able quan i y o biomolecules o
he apeu ic d ugs, acili a ing hei con olled elease.
29,30
The composi ion o SiO
2
-CaO nanopa icles is conduci e o
p omo ing os eo egene a ion.
31
In ecen yea s, he e has
been significan wo k in he field o ad anced bioinks o
bio ab ica ion, including he inco po a ion o phy o he -
apeu ic agen s and ino ganic fille s in o hyd ogel bioinks.
Se e al esea ch g oups ha e success ully demons a ed he
e ficacy o enhancing ADA-GEL hyd ogels h ough he
inco po a ion o bioac i e glass nanopa icles. Fo example,
Sa ke e al.
32
demons a ed ha he inco po a ion o silica e
Bide e al. 525
bioac i e glass in o ADA-GEL may imp o e he me-
chanical s eng h o sca olds.
32
Lei e e al.
24
u ilized 3D
biop in ing o c ea e ADA-GEL cons uc s ein o ced
h ough he inco po a ion o bioac i e glass nanopa icles.
These nanopa icles we e ound o induce he o ma ion o
an apa i e laye , mimicking he na i e bone s uc u e upon
imme sion in o a simula ed body fluid (SBF).
24
Mona a i
e al.
33
c ea ed 3D p in ed ADA-GEL sca olds wi h bio-
ac i e glass pa icles loaded wi h ica iin, which we e able o
inc ease he ac i i y o os eoblas cells, esul ing in im-
p o ed cell p oli e a ion, adhesion, and di e en ia ion.
33
In
his s udy, ou p ima y ocus was on examining he im-
p o emen s ha should esul om he simul aneous in-
clusion o FA and MBGNs (in wo di e en concen a ions)
wi hin ADA-GEL hyd ogel. Mo e p ecisely, we assessed
he e ec on deg ada ion and swelling beha iou , as well as
mechanical deg ada ion h ough comp ession es s. Addi-
ionally, we in es iga ed he elease p ofiles o FA, Ca, and
GEL om ADA-GEL-based samples. Since he in ended
applica ion o he composi e hyd ogels is in he field o 3D
(bio)p in ing, we pe o med deg ada ion/swelling and elease
s udies wi h 3D p in ed sca olds (in addi ion o cas films). To
assess he sui abili y o ou inks o 3D (bio)p in ing, we
conduc ed heological measu emen s and 3D p in ing expe -
imen s on all inks. In addi ion o he filamen usion es (FFT),
filamen collapse es (FCT), and g id s uc u e es (GST), we
p in ed complex s uc u es, including highe and la ge sca -
olds, bone-shaped and s a -shaped designs, using he mos
p omising inks. Fu he mo e, o explo e he impac o FA and
MBGNs on Schi ’s base o ma ion in ADA-GEL, we pe -
o med a deg ee o c osslinking es . To demons a e he
possible mine aliza ion esul ing om he MBGN con en ,
bioac i i y es s we e ca ied ou by imme sing samples in a
SBF solu ion o 28 days. In i o cy ocompa ibili y s udies
we e also conduc ed wi h he p e-os eoblas ic cell line
MC3T3-E1 on all inks. Addi ionally, we examined he elease
o ascula endo helial g ow h ac o -A (VEGF-A), p o iding
ini ial insigh s in o he dual e ec o FA and MBGNs on ADA-
GEL bioinks p ope ies.
Ma e ials & me hods
Ma e ials
GEL om po cine skin (Type A, gel s eng h 300 bloom),
comme cially a ailable high pu i y (pha maceu ical g ade)
algina e acid powde (sodium sal om ma ine b own algae,
Vi apha m, Ge many), calcium chlo ide dehyd a e (CaCl
2
),
sodium (me a)pe ioda e (NaIO
4
), e hylene glycol and ans-
FA ( ans-4-Hyd oxy-3-me hoxycinnamic acid, 99%), so-
dium hyd ogen ca bona e (NaHCO
3
), and ini-
obenzenesul onic acid (TNBS) we e pu chased om
Sigma-Ald ich (Ge many). Chemicals used o he sol-gel
syn hesis o MBGNs: ce yl ime hylammonium b omide
(CTAB), e hyl ace a e (EA), e ae hyl o hosilica e
(TEOS, ≥99%), and calcium ni a e e ahyd a e (CN)
(Ca(NO
3
)
2
4H
2
O, ≥99%), we e pu chased om Sigma-
Ald ich (Ge many). Mo eo e , ammonium hyd oxide so-
lu ion (NH
4
OH, 28% basic solu ion) and e hanol (99.8%)
we e pu chased om VWR In e na ional (Ge many).
Dulbecco´s Phospha e Bu e ed Saline (DPBS, [-] Ca
2+
, [-]
Mg
2+
), Hank’s Balanced Sal Solu ion (HBSS); Calcein AM
and 40,6-Diamidin-2-phenylindol (DAPI), alpha-Modified
Eagle’s medium (α-MEM) wi hou ibonucleosides and
depxy ibonucleosides, penicillin-s ep omycin (PS) and
L-Glu amine, we e ob ained om The moFishe , In i ogen
(Ge many). Mic obial ansglu aminase (mTG) was ob-
ained om Ajinomo o Foods, Eu ope.
ADA syn hesis
The ADA used in his s udy was syn hesized ollowing a
p ocedu e desc ibed o iginally by Sa ke e al.
34
B iefly,
10 g o sodium algina e we e suspended in 10 mL o
e hanol. Following his, 1.337 g o NaIO
4
we e dissol ed in
50 mL o MilliQ wa e , and he esul ing solu ion was added
o he algina e-e hanol suspension. This mix u e was s i ed
o a du a ion o 6 h subsequen ly being quenched wi h
10 mL o e hylene glycol, ollowed by an addi ional 30 min
o s i ing. A e 30 min, he s i ing was s opped o acili a e
he sedimen a ion o he ADA. The esul an ma e ial un-
de wen a 3 day p ocess o dialysis agains MilliQ wa e and
was subsequen ly eeze-d ied.
Syn hesis o MBGNs
MBGNs wi h a nominal composi ion o 70% SiO
2
and 30%
CaO (mol%) we e syn hesized using a mic oemulsion based
sol-gel me hod ollowing he app oach epo ed else-
whe e.
35
Fo his, 2.8 g CTAB was dissol ed in 132 mL
deionized wa e a 35°C. Subsequen ly, EAwas added while
lowe ing he empe a u e o 25°C o 30 min. A e 30 min
o s i ing 28 mL o 1 M aqueous NH
4
OH was added and
s i ed o 15 min. Then, 14.4 mL TEOS was added and
s i ed o 30 min, ollowed by he addi ion o 9.12 g o CN.
The solu ion was s i ed o 4 h. A e 4 h, he pa icles we e
collec ed using a cen i uge, washed 3 imes wi h deionized
wa e and once wi h e hanol (99.8%). The pa icles we e le
o d y o e nigh in an o en a 60°C, a e which hey we e
calcina ed a 700°C o 5 h and 35 min wi h a hea ing a e o
2°C/min. The empe a u e was kep cons an o ano he 3 h.
Hyd ogel p epa a ion
To o mula e ADA-GEL, 5% (w/ ) lyophilized ADA was
dissol ed a RT and 7.5% (w/ ) GEL a 37°C in DPBS. The
solu ions we e mixed in 1:1 a io, see Figu e 1 (1A and 2) o
10 min o c ea e 2.5%ADA-3.75%GEL. Fo ADA-GEL-FA
526 Jou nal o Bioma e ials Applica ions 39(6)
samples he FA concen a ion was se o 0.15% (w/ ) based on
ou p e ious esul s.
20
B iefly, 0.3% (w/ ) FA powde was
dissol ed in DPBS a 60°C o 2–3 h unde con inuous s i ing
un il i was ully dissol ed. Subsequen ly, ADA was added o
he FA-DPBS solu ion and s i ed un il achie ing a homog-
enous solu ion. Las ly, GEL was added o he ADA-FA so-
lu ion (Figu e 1 (1B and 2)) o c ea e 2.5%ADA-3.75%GEL-
0.15%FA hyd ogel p ecu so . To p epa e ADA-GEL-MBGN
o ADA-GEL-FA-MBGN hyd ogels, 0.1% (w/ ) and 0.5%
(w/ ) o MBGNs we e added o ADA o ADA-FA (see
Figu e 1 (1C)). To ensu e he bes dis ibu ion o pa icles, he
mix u e was s i ed o 10 min and placed in o an ul asonic
ba h o 30 min. This s ep was epea ed wice. When pa icles
dis ibu ion was achie ed, GEL was added o he ADA-
MBGNs o ADA-FA-MBGNs mix u e (see Figu e 1 (1C
and 2)). All used inks in his s udy a e lis ed in Table 1.
Fab ica ion o films
In Figu e 2(a), a schema ic illus a ion o he p epa a ion o
ADA-GEL-based films is shown. In o de o p epa e ADA-
GEL films wi h/wi hou FA and MBGNs, he hyd ogel
solu ion (p epa a ion desc ibed in he p e ious sec ion) was
added in o p e-cooled cylind ical silicon moulds wi h a
diame e o 12 mm and a heigh o 2 mm. An excep ion in
ega d o size was made o he mic o es e comp ession
measu emen s, whe e a silicon mould wi h a diame e o
5 mm and a heigh o 2 mm was used. To ensu e he he mal
gela ion o GEL all moulds we e placed in o a 4°C idge o
10 min. Subsequen ly, he hyd ogel films we e c osslinked
wi h a c osslinking solu ion (2.5% (w/ ) mTG in a 0.1 M
CaCl
2
) o 10 min. Du ing he c osslinking ime, he films
we e de ached om he silicon mould wi h a spa ula. Fo
s e ile composi e films p epa a ion, MBGNs we e hea
s e ilized o 2 h a 160°C p io o mixing and all hyd ogels
we e s e ilized by s e ile fil a ions wi h 0.22 µm ( o GEL)
and 0.45 µm ( o ADA and ADA-FA) po e size memb ane
fil e s unde s e ile condi ions.
Fab ica ion o 3D cons uc s/3D p in ing
In Figu e 2(b) he ab ica ion o 3D p in ed cons uc s wi h
all hyd ogels is displayed. B iefly, all inks we e ans e ed
in o ca idges p io o p in ing, which we e placed in o a
4°C idge o 5 –10 min o achie e a sui able iscosi y o
GEL (due o he gela ion p ocess o GEL). The inks we e
Figu e 1. Schema ic illus a ion o he p epa a ion o ADA-GEL-based hyd ogels.
Bide e al. 527
ex uded wi h a 3D p in e (BioSca olde 3.1, GeSim) laye
by laye o c ea e 4-laye ed g id-like s uc u es using a nozzle
wi h a diame e o 250 µm and a p in ing speed o 7 mm/s.
All p in ing pa ame e s we e adjus ed wi h he g id s uc u e
es (GST, desc ibed in de ail u he below) and kep cons an
h oughou he sample p epa a ion.
Physicochemical cha ac e iza ion
Scanning elec on mic oscopy (SEM, AURIGA®, Zeiss)
images o MBGNs we e ob ained o cha ac e ize he
mo phology, dis ibu ion, and size o MBGNs. Fo SEM
obse a ion, pa icles we e added homogenously on op o a
ca bon ape. To in es iga e he elemen al composi ion o
MBGNs ene gy dispe si e spec oscopy (EDS, X-MaxN
Ox o d Ins umen s, UK) was pe o med using 10 kV
ol age and a 6 mm wo king dis ance du ing SEM obse -
a ions. Mo eo e , ansmission elec on mic oscopy
(TEM, TALOS F200S, The mo Fische Scien ific, USA)
was used o obse e he mic os uc u e o MBGNs.
Mo eo e , he MBGNs we e cha ac e ised using an ART-
FTIR spec oscopy (IRA fini y-1S, Shimadzu, Ge many) in
o de o in es iga e he su ace chemis y. The measu e-
men s we e pe o med in abso bance mode om wa e-
numbe 4000 o 400 cm
1
by applying 40 scans wi h a
esolu ion o 4 cm
1
.
In i o deg ada ion/swelling beha iou
A deg ada ion/swelling s udy was conduc ed o examine he
influence o FA and MBGNs on such beha iou . ADA-GEL
films and/o 3D p in ed sca olds (N= 6, espec i ely) o all
Table 1. Composi ion and designa ion o all used inks.
ADA (w/ ) (%) GEL (w/ ) (%) FA (w/ ) BG (w/ ) Designa u e
2.5 3.75 - - ADA-GEL
2.5 3.75 0.15% ADA-GEL-FA
2.5 3.75 - 0.1% ADA-GEL-0.1%MBGN
2.5 3.75 - 0.5% ADA-GEL-0.5%MBGN
2.5 3.75 0.15% 0.1% ADA-GEL-FA-0.1%MBGN
2.5 3.75 0.15% 0.5% ADA-GEL-FA-0.5%MBGN
Figu e 2. (a) Schema ic illus a ion o he p epa a ion o ADA-GEL-based films. (b) Schema ic illus a ion o he p epa a ion o ADA-
GEL-based 3D p in ed sca olds.
528 Jou nal o Bioma e ials Applica ions 39(6)

composi ions (lis ed in Table 1) we e assessed o hei
weigh changes o e a 28 day incuba ion pe iod in cell
cul u e medium. B iefly, s e ile condi ions we e main ained
while p epa ing films wi h dimension o 12 mm wide and
2 mm heigh and/o 1 × 1 cm
2
3D p in ed sca olds o each
composi ion. All samples we e placed in o p e-weighed
inse s, which we e hen posi ioned in a 6-well pla e and
co e ed wi h cell cul u e medium. The samples we e s o ed
unde cell cul u e condi ions a 37°C and 5% CO
2
wi h he
medium e eshed wice a week. Weigh measu emen s
we e aken wi hin he fi s 3 h a 15-min in e als, and
subsequen ly a e 24 h, 3, 7, 14, 21, and 28 days. The
pe cen age change in weigh (W
) was calcula ed using he
ollowing equa ion:
W ¼W W0
W0

× 100 (1)
whe e W
0
ep esen s he ini ial weigh o he samples be o e
imme sion in he cell cul u e medium, and W
deno es he
cu en weigh o he samples a e imme sion in o he cell
cul u e medium.
D ug elease s udies
FA elease. The impac o he MBGN con en on FA elease
om ADA-GEL-based films and/o sca olds was de e -
mined by a FA elease s udy. B iefly, 6 s e ile ADA-GEL-
FA, ADA-GEL-FA-0.1%MBGN and ADA-GEL-FA-0.5%
MBGN films and 1 × 1 cm
2
3D p in ed sca olds we e
p epa ed and p in ed, espec i ely, loca ed in o 6-well pla es
and co e ed wi h HBSS. ADA-GEL-FA samples we e used
as e e ence ma e ial. The FA elease was measu ed cu-
mula i e ia UV-Vis spec ome e (Speco d40, Analy ic
Jena, Ge many) a a wa eleng h o 310 nm and using he
so wa e WinASPECT 2.5.8.0. The abso bances we e
measu ed a e 24 h, 3, 7, 14, 21 and 28 days o incuba ion.
The e o e, a each ime poin 1 mL o HBSS supe na an
was collec ed o he measu emen and eplaced wi h esh
HBSS. The FA elease was measu ed in µg/µl.
Ca elease. The calcium elease ou o films and 3D p in ed
sca olds was measu ed wi h a calcium colo ime ic assay
ki (MAK022, Sigma-Ald ich, Ge many). Wi h his ki , he
calcium ion concen a ion can be measu ed based on
ch omogenic complexes o calcium ions a aching o
o-c esolph halein, which is p opo ional o he concen a-
ion o calcium ion. B iefly, 90 µl o ch omogenic eagen
and 60 µl calcium assay bu e we e added o a 96-well pla e
con aining 50 µl o sample. The expe imen was ca ied ou
a RT and unde he absence o ligh , due o he ligh
sensi i i y o he assay. A e 5 –10 min incuba ion a RT a
colou change o iole occu ed he ea e he abso bance
was measu ed a a wa eleng h o 575 nm ia a mic opla e
eade (PHOmo eade , Au obio Diagnos ics Co., L d). The
concen a ion o calcium was calcula ed using he ollowing
equa ion:
Concen a ion o calcium ¼Sa
S
(2)
whe e S
a
is he unknown calcium amoun [µg] aken ou o
he p io p epa ed s anda d cu e and S
is he olume o he
samples, which is in his case 50 µl.
GEL elease. To in es iga e he GEL elease ou o films and
3D p in ed sca olds a colo ime ic p o ein assay, such as he
Coomassie blue G-250 dye-binding (B ad o d) assay was
ca ied ou . The B ad o d p o ein assay is a equen ly used
colo ime ic me hod o de ec he p o ein (GEL) concen-
a ion. The e o e, a calib a ion cu e was p epa ed p io he
expe imen wi h known GEL concen a ions. By mixing he
samples wi h B ad o d solu ion a colou eac ion change o
blue occu s. The in ensi y o he colou change is p opo -
ional o he con en o a oma ic and basic amino acids. The
op ical abso bance was measu ed by UV-Vis spec oscopy
a a wa eleng h 595 nm.
Deg ee o c osslinking
In o de o in es iga e he impac o MBGNs on he Schi ’s
base o ma ion o ADA-GEL-based hyd ogels, a ini-
obenzenesul onic acid (TNBS) assay was pe o med. This
expe imen allows he de e mina ion o ee amino acids
(NH
2
-g oups) o GEL. I ADA ( ee aldehyde g oups) is
p esen , a e e sible Schi ’s base o ma ion occu s which
educe he p esence o NH
2
-g oups in ADA-GEL samples.
As e e ence ma e ials 3.75% (w/ ) GEL samples (posi i e
con ol) and pu e ADA 2.5% (w/ ) samples (nega i e
con ol) we e used. The assay was conduc ed ollowing
p e ious wo k epo ed by Nguyen e al.
36
B iefly, ADA,
GEL, ADA-GEL, ADA-GEL-0.1%MBGN, ADA-GEL-
0.5%MBGN, ADA-GEL-FA-0.1%MBGN and ADA-GEL-
FA-0.5%MBGN hyd ogels we e p epa ed, ozen and
eeze d ied (N= 5 espec i ely). Subsequen ly, 5 mg o he
lyophilized ma e ial was sol ed in 1 mL o 4% solu ion o
NaHCO
3
a 60°C wi h he help o a o ex mixe . Nex ,
1 mL o a 0.5% ( / ) solu ion o yellow TNBS was added o
each sample and o exed again. A blank sample was used
as a e e ence du ing he UV-Vis measu emen s. Subse-
quen ly, samples we e s o ed in a shaking incuba o o 4 h
and 60°C. Wi hin he 4 h pe iod, a colou change om
yellow o o ange occu ed. A e incuba ion, 1 mL o each
solu ion was added o 3 mL o 6 M HCL solu ion and
incuba ed in he shaking incuba o o u he 1.5 h a 40°C.
Du ing he 1.5 h pe iod, he colou changed om o ange o
yellow. Las ly, 1 mL o each sample was ans e ed in o
UV-cu e es, whe eas he blank samples we e se as he
Bide e al. 529
e e ence ma e ial. The measu ed abso bances we e de-
ec ed by a UV-Vis spec ome e a 346 nm. I is known ha
he highe he abso bances he highe is he amoun o ee
NH
2
-g oups and he lowe is he deg ee o c osslinking (%
D
c
). The %D
c
was ecalcula ed using he ollowing
equa ion:
%Dc¼Aunc osslinked Ac osslinked
Aunc osslinked
× 100 (3)
HAp o ma ion
The HAp o ma ion abili y on he su ace o ADA-GEL
samples con aining MBGNs was assessed by conduc ing a
bioac i i y s udy o e 28 days o imme sion in a simula ed
body fluid (SBF) solu ion.
37
Films we e imme sed wi hin a
ce ain amoun o SBF solu ion and incuba ed a 37°C wi h
con inuous shaking (80 pm). The SBF solu ion was
changed wice a week o simula e condi ions wi hin he
human body and o main ain he ionic concen a ion wi hin
he SBF solu ion. Fou eplica es we e p epa ed o each
ime poin . ADA-GEL-0.1%MBGN, ADA-GEL-0.5%
MBGN, ADA-GEL-FA-0.1%MBGN and ADA-GEL-FA-
0.5%MBGN hyd ogels we e used o ab ica e films o di-
mensions 12 mm × 2 mm. Films we e aken ou o he SBF
solu ion (25 mL) a e 1, 3, 7, 14, 21 and 28 days and insed
wi h dis illed wa e . Two o hese ou ADA-GEL-based
eplica es wi h/wi hou FA and wi h/wi hou MBGNs we e
p epa ed o SEM using a SEM-FIX I and SEM-FIX II so-
lu ion, composed o 0.1% glu a aldehyde and 2% pa a o -
maldehyde and 0.3% glu a aldehyde and 3%
pa a o maldehyde, espec i ely, each o 1 h. A e wa ds,
samples we e imme sed in an e hanol se ies ( om 30% up o
99%) o 30 min each. Subsequen ly, he samples we e d ied
using a c i ical poin d ye (EM CPF3000, Leica) and fixed
on o a ca bon ape o SEM obse a ion. The o he wo o he
ou eplica es we e eeze d ied ( eeze d ye , ALPHA 1-
2 LDplus, CHRIST, Ge many). In o de o in es iga e he
HAp o ma ion on he su ace o samples he eeze d ied
samples we e analysed using ATR-FTIR spec oscopy in
abso bance mode om wa enumbe 4000 o 400 cm
1
wi h a
esolu ion o 4 cm
1
. XRD (MiniFlex 600, Rigaku) was also
ca ied ou o ack possible changes in he c ys alline phases.
The XRD analysis was measu ed be ween 2θ20° and 80°
wi h a esolu ion o 0.02° and a scanning a e o 4°/min.
Mo eo e , SEM obse a ions and EDS analysis we e pe -
o med in o de o assess he su ace mo phology.
De e mina ion o ion elease om MBGNs in SBF
The elease o Si and Ca ions was measu ed by induc i ely
coupled plasma-op ical emission spec ome y (ICP-OES,
Agilen 5100 SVDV). B iefly, MBGNs in concen a ion o
0.1% (w/ ) and 0.5% (w/ ) we e imme sed in o 30 mL o
SBF solu ion in alcon ubes. Tubes we e incuba ed a 37°C
in a shaking incuba o (KS 4000 i con ol, IKA, Ge many)
a 80 pm. A e 7, 14 and 21 days o incuba ion in SBF,
alcon ubes we e cen i uged a 7830 pm o 15 min and
supe na an s we e collec ed. Supe na an s we e s abilized
wi h concen a ed HNO
3
un il a pH o ≤2 was eached and
fil e ed using a 0.22 µm fil e . Pu e SBF solu ion wi hou
any MBGNs was used as e e ence solu ion. P io o he
analysis, h ee calib a ion solu ions we e o mula ed o
es ablish a linea ela ionship be ween in ensi y and con-
cen a ion. These calib a ion solu ions we e c ea ed using
e e ence s anda ds ha a e ce ified o ICP echniques. The
ime depended concen a ion elease o Ca and Si ions in o
SBF medium was measu ed in cumula i e o m.
Mechanical cha ac e iza ion
Comp ession es . To de e mine he e ec i e modulus o
films and o in es iga e he impac o MBGNs on me-
chanical p ope ies, a es was pe o med in comp ession
using a Mic o es e (Mic o es e LT, Cellscale). Samples
(N= 4 pe composi ion, compa e Table 1) wi h a diame e o
5 mm and heigh o 2 mm we e p epa ed in s e ile con-
di ions, placed in o a 24-well pla e, co e ed wi h cell cul u e
medium and s o ed a 37°C and 5% CO
2
humidi y. The
medium was changed 2 imes a week. The measu emen s
we e pe o med a e 24 h, 3, 7, 14, 21 and 28 days. Day
0 samples we e measu ed di ec ly a e c osslinking and
wi hou any imme sion in cell cul u e medium. The
c osslinking solu ion was composed o 2.5% (w/ ) mTG in
0.1 M CaCl
2
solu ion. Fo he measu emen a 6 mm × 6 mm
pla e and a can ile e beam wi h a diame e o 0.5588 mm
we e used. The es was se o 20% o displacemen wi h
4 cycles pe measu emen . Each cycle consis ed o 20 s
loading ime, 2 s o holding ime and a eco e y o 20 s wi h
afinal holding ime o 2 s. The e ec i e modulus o each
sample was calcula ed by he slope o he 5% –10% sec ion
o each ob ained s ess-s ain cu e. The o ce and maxi-
mum comp ession s ess alues we e ex ac ed om he
ob ained da a.
38,39
Rheological e alua ion. Rheological measu emen s we e
used o in es iga e he impac o MBGNs on he iscosi y o
ADA-GEL-based hyd ogels and he e o e o assess he
shape fideli y o he 3D p in ed sca olds du ing he 3D (bio)
p in ing p ocess. All inks men ioned in Table 1 we e used
o he iscosi y e alua ion. Fo he measu emen s a dis-
co e y HR 3 heome e (Disco e y Hyb id Rheome e s, TA
ins umen s, USA) was used. This de ice is equipped wi h a
cone pla e geome y (cone o 2°). P io o he measu e-
men s, an ampli ude sweep was pe o med wi h pu e ADA-
GEL as a e e ence o adjus he linea iscoelas ic egion.
Subsequen ly, equency sweep es s we e pe o med o all
530 Jou nal o Bioma e ials Applica ions 39(6)
inks. All composi ions we e measu ed 3 imes a RT. B iefly,
100 µl o each hyd ogel was added o he lowe pla e o he
heome e . The ampli ude sweep was pe o med wi h a
cons an equency o 10 ad/s wi h a s ain ange o 1 o
200%. The equency sweep was conduc ed in a equency
ange o 0.1 o 100 Hz and a a cons an elonga ion os-
cilla ion s ain o 10%, which was p io de e mined.
Assessmen o 3D p in abili y
The capabili y o hyd ogels o be 3D p in ed was examined
h ough a ious expe imen s conduc ed wi h a 3D p in e
(BioSca olde 3.1, GeSim). All inks (see Table 1) we e
p epa ed as men ioned abo e. ImageJ so wa e was used o
e alua e he esul s o all 3D p in ing expe imen s. Figu e 3
illus a es he pe o med 3D p in ing expe imen s, which a e
explained in de ail below.
Filamen usion es (FFT). The FFT was conduc ed o de-
e mine he achie able esolu ion o di e en inks. This
pa ame e can be assessed as he minimum dis ance a
which wo adjacen s ands me ge. The es was inspi ed by
he wo k o Ribei o e al.
40
The expe imen in ol ed
p in ing a one-laye ed meande ing pa e n wi h g adually
educing dis ance be ween he s ands, demons a ing he
poin a which wo adjacen s ands become indis in-
guishable. In Figu e 3(a) a p in ed s uc u e is depic ed wi h
a g adually changing dis ance be ween he s ands, a ying
om 2, 1.5, 1, 0.75 and o 0.5 mm. The a io be ween he
used segmen leng h (
s
) and he filamen hickness (
) was
e alua ed and plo ed agains he filamen dis ance (
d
), as
commonly epo ed in he li e a u e.
41
Filamen collapse es (FCT). The FCT p o es aluable in
de e mining he maximum dis ance be ween wo objec s
whe e a p in ed s and emains in ac wi hou collapsing.
This expe imen is based on he idea in oduced by Ribei o
e al.
40
and The iaul e al.
42
This es is pa icula ly c ucial
o po ous s uc u es, ensu ing ha s ands o e po es do no
ea down. To conduc he es , he ink is p in ed o e a
s uc u e ea u ing pilla s posi ioned a inc easing dis ances,
as illus a ed in Figu e 3(b). The deflec ion angle was as-
sessed o h ee samples o each composi ion using ImageJ
so wa e.
G id s uc u e es (GST). The idea o he GST was acqui ed
om p e ious wo k epo ed by Hazu e al.
43
B iefly, a 1 ×
1cm
2
g id (see Figu e 3(c)) composed o ou laye s was
p in ed. The p essu e was kep cons an in he ange o 15 ±
10 kPa in o de o compa e he geome y o s ands and
po es o all p in ed composi ions. All 3D p in ed sca olds
we e imaged wi h a da k field mic oscope (S emi 508, Ca l
Zeiss, Ge many). The mic oscopy images we e e alua ed
using ImageJ, whe e calcula ions we e pe o med o
de e mine he s and wid h e ically (s
) and ho izon ally
(s
h
), as well as he po e wid h e ically (w
) and ho i-
zon ally (w
h
) (ma ked in Figu e 3(c)). Each 3D p in ed
sca old was measu ed fi e imes wi hin he same image.
The ideal diagonal o a po e was hen es ablished by he
ollowing equa ion:
dideal ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
w2
þw2
h
q(4)
This alue was hen compa ed wi h he ac ual diagonal
(d
m
) using he diagonal co ela ion a e (DCR):
DCR ¼dm
dideal
(5)
Complex s a -shape p in ing. Complex s uc u e p in ing was
pe o med o ADA-GEL-FA and ADA-GEL-FA-0.1%
MBGN, which we e he mos p omising (bio)inks (see he
Resul s sec ion below). In pa icula , s a -shaped s uc u es
we e p in ed as desc ibed by Wu e al.
44
The quan i a i e
es in ol es he e alua ion o he angula de ia ion (D
a
)
be ween he heo e ical angle (θ
) (acu e and ob use) and he
expe imen al angle (θ
e
) (acu e and ob use) measu ed using
ImageJ:
Da¼θeθ
θ
(6)
The expe imen al angle (θ
e
) was calcula ed o bo h acu e
and ob use angles by d awing a line connec ing he e ex o
he in e sec ion (see Figu e 3(d)). The heo e ical angle (θ
)
was se a 36° o he acu e angle and 108° o he ob use
angle.
44
In i o cy ocompa ibili y
In o de o analyse he impac o MBGNs and FA on cell
iabili y, a cy o oxici y es was pe o med. Fo his pu -
pose, he undi e en ia ed p eos eoblas ic cell line MC3T3-
E1 was used. Pu e ADA-GEL hyd ogel was used as a
e e ence ma e ial. Fo all hyd ogel composi ions (see
Table 1)1×10
6
cells/ml we e inco po a ed. A e he
sample p epa a ion (films and/o 3D biop in ed sca olds),
all samples we e c osslinked wi h a c osslinking solu ion
composed o 2.5% (w/ ) mTG in 0.1 M CaCl
2
solu ion o
10 min. The ea e , all samples we e co e ed wi h α-MEM
cell cul u e medium (de ails men ioned below) and s o ed in
an incuba o unde cell condi ions.
Di ec cell cul u e es . In o de o pe o m di ec cell cul u e
es s, 50 µl o each MC3T3-E1 cell laden hyd ogel was
pipe ed in o each well o a 48-well pla e, c osslinked,
washed and co e ed wi h cell cul u e medium un il u he
s ainings (Calcein AM and DAPI). S ainings we e
Bide e al. 531
pe o med a e 24 h and 7 days o incuba ion. Cell cul u e
medium was changed wice a week. Pu e ADA-GEL
samples wi hou any cells we e p epa ed as e e ence.
3D biop in ing. Fo he 3D biop in ing p ocess, all cell laden
ADA-GEL hyd ogels (lis ed in Table 1) we e ans e ed in o
ca idges and ins alled in o he GeSim 3D p in e desc ibed
abo e. Subsequen ly, 4 laye s sca olds wi h a size o 1 ×
1cm
2
we e p in ed in o 6-well pla es (N=6).
Cell cul u e. Fo he p eos eoblas ic cell line MC3T3-E1 a
α-MEM medium wi h 1.0% ( / ) PS, 1.0% ( / )
L-Glu amine and wi h 10% ( / ) o Fe al Bo ine Se um
(FBS, Sigma-Ald ich, Tau ki chen, Ge many) was needed.
Cells we e incuba ed a 37°C, 5% CO
2
and 95% ela i e
humidi y, and we e spli ed/used o expe imen s a e
eaching a confluency o 80% –90%.
Cell iabili y. The iabili y o MC3T3-E1 cells embedded in
all es ed inks was de e mined by using a WST-8 cell
coun ing ki (Cell-Couning-Ki 8, Sigma-Ald ich,
Ge many). The eby, a 5% ( / ) WST-8 solu ion was used.
The abso bances we e measu ed a e 4 h o incuba ion wi h
a pla e eade (PHOmo mic opla e eade , China) a a
wa eleng h a 450 nm. The ac i i y o cells co ela es wi h
he in ensi y o he measu ed abso bance h ough a cali-
b a ion cu e.
Li e/dead s aining. Subsequen o he WST-8 es , a li e/dead
s aining was ca ied ou . Fo he li e s aining Calcein AM
(In i ogen, Ge many) was used, whe eas o dead cell
s aining DAPI (In i ogen, Ge many) was used. Calcein
AM is a s aining which is used o in es iga e he amoun o
li ing cells and DAPI is used o assess he nuclei o cells
esul ing in he o al amoun o cells. Mo e de ailed, a e
disca ding he WST-8 solu ion and a subsequen washing
s ep, he Calcein AM solu ion in a 4 µl/ml concen a ion in
HBSS was added o he samples o 45 min in da k con-
di ions. A e 45 min incuba ion ime, he Calcein AM
solu ion was disca ded, washed and fixed wi h a 3.7%
o maldehyde solu ion in HBSS o 10 min. A e he
fixa ion, a 1 µl/ml DAPI solu ion in HBSS was added o he
Figu e 3. Schema ic illus a ion o 3D p in ing expe imen s. (a) Illus a ion o FFT pa e n. (b) The pla o m employed o he FCT
ea u ing pilla s wi h p og essi ely inc easing dis ances and showcasing he deflec ion angle o a p in ed filamen na iga ing wo
obs acles. (c) GST, x was se o 5 measu emen s. (d) Complex s a -shape p in ing and expe imen al angles o calcula ing he angula
de ia ion.
532 Jou nal o Bioma e ials Applica ions 39(6)
was in a highe ange han in he p esen s udy, which migh
be due o he addi ional enzyma ic c osslinking wi h mTG
and he addi ional c osslinking o FA o GEL p esen in ou
s udy.
15,21
Mo eo e , la ge sca olds we e p in ed in hei
wo k, esul ing in mo e ma e ial p esen and hence highe
GEL elease.
33
Deg ee o c osslinking
In ou p e ious wo k he impac o FA on he Schi ’s base
o ma ion in ADA-GEL was al eady shown
20
. I is appa en
ha he highes concen a ion o 0.5% (w/ ) MBGNs wi hin
ADA-GEL-based hyd ogels migh dis up he hyd ogel
ne wo k leading o in e up ion o he Schi ’s base o -
ma ion esul ing in a as e deg ada ion o samples, as e
FA elease and accele a ed GEL elease, indica ing ha a
deg ee o c osslinking es is o he highes impo ance. To
de e mine he e ec o MBGNs on he e e sible c oss-
linking o ADA and GEL, pu e GEL was used a e e ence.
Figu e 7( ) shows he highes deg ee o c osslinking (18%)
o ADA-GEL-FA samples confi ming ha FA c osslinks o
GEL esul ing in less ee NH
2
-g oups and hus highe
deg ee o c osslinking. The ADA-GEL deg ee o c oss-
linking was 13%. ADA-GEL and ADA-GEL-0.1%MBGN
eached he same ange o c osslinking deg ee, indica ing
ha a concen a ion o 0.1% (w/ ) MBGNs does no hinde
o in e up he Schi ’s base o ma ion. ADA-GEL-FA-
0.1%MBGN samples eached sligh ly highe deg ee o
c osslinking (15%), which migh be again an indica ion o
he addi ional c osslinking be ween FA and GEL, howe e ,
no significan ly di e en . In e es ing is ha ADA-GEL-
0.5%MBGN eached he lowes deg ee o c osslinking wi h
11% indica ing ha a high MBGNs con en migh dis up
he hyd ogel ne wo k esul ing in a dis up ion o Schi ’s
base o ma ion and hus a lowe deg ee o c osslinking.
Compa ing ADA-GEL-0.5%MBGN wi h ADA-GEL-FA-
0.5%MBGN one can obse e a highe deg ee o c oss-
linking o FA con aining samples which again confi ms he
addi ional c osslinking o FA and GEL. ADA-GEL-FA and
ADA-GEL-FA-0.1%MBGN samples appea ed o ha e he
highes deg ee o c osslinking, especially no able du ing 3D
p in ing, esul ing in high esolu ion and high shape fideli y
o 3D p in ed sca olds wi h hese wo inks (see 3D p in ing
sec ion).
HA o ma ion
The o ma ion o a HAp laye on he su ace o MBGN
con aining samples demons a es hei bioac i e na u e and
hei po en ial o p omo ing os eoconduc i i y.
68
To con-
fi m he bioac i i y o MBGNs and in es iga e he impac o
FA on his p ope y, a bioac i i y s udy was pe o med.
B iefly, he hyd ogel films o ADA-GEL-0.1%MBGN,
ADA-GEL-0.5%MBGN, ADA-GEL-FA-0.1%MBGN and
ADA-GEL-FA-0.5%MBGN we e imme sed in SBF o
28 days. The confi ma ion o HAp o ma ion was analysed
by SEM-EDS (Figu e 6(a)), ART-FTIR (Figu e 6(b)) and
XRD (Figu e 6(c)) analyses. SEM-EDS analysis was used
o isualize he ADA-GEL composi e films and de e mine
he elemen al composi ion o HAp. A e 28 days he el-
emen al cha ac e iza ion h ough EDS analysis confi med
he o ma ion o HAp o ADA-GEL-0.1%MBGN wi h Ca/
P = 1.32 (a omic%), o ADA-GEL-0.5% wi h Ca/P = 1.36
(a omic%), o ADA-GEL-FA-0.1%MBGN wi h Ca/P =
1.54 (a omic%) and ADA-GEL-FA-0.5%MBGN wi h Ca/
P = 1.56 (a omic%). Acco ding o li e a u e HAp wi h a
calcium deficiency, deno ed as Ca
10x
(PO
4
)
6
x (HPO
4
)x
(OH)
2x
, whe e 0 ≤x≤1, is o highe biological signifi-
cance compa ed o s oichiome ic HAp. This in e es a ises
om he ac ha he Ca/P a io in bone closely app oxi-
ma es 1.5.
69–71
The ideal Ca/P a io in a omic% o HAp
wi h he composi ion Ca(PO
4
)
3
OH is 1.67. This ideal a io
is a he di ficul o achie e, since his alue e e o a EDX
measu emen wi h a fla su ace, which migh no be he
case o ADA-GEL samples a e c i ical poin d ying.
Mo eo e , due o he p esence o phospho (P) in he SBF
solu ion he ac ual P con en migh be highe esul ing in a
deficiency o Ca ions. The e o e, in his s udy, he EDX
analysis should simply gi e a ough o e iew abou he
p esence o Ca and P. To iden i y he o ma ion o HAp on
he su ace o he sample mo e p ecisely FTIR and XRD
measu emen s we e pe o med. In Figu e 6(b), in all es ed
composi ions sha p peaks a 1038 cm
1
o 1020 cm
1
a e
isible, which indica e PO
4
3-
s e ching and is he e o e an
indica ion o HAp. This peak becomes mo e p ominen
a e 14 days o incuba ion in SBF.
68,72
The peak a
875 cm
1
s ands o CO
3
2-
ib a ion.
72
Mo eo e , he
shoulde s a 875 cm
1
and 959 cm
1
migh be associa ed
wi h HAp.
68,72
In all composi ions, peaks a 560 cm
1
and 600 cm
1
we e e iden and can be associa ed o he
P-O bond in c ys allized calcium phospha e, indica ing
he o ma ion o a CaP phase.
9,72,73
All men ioned peaks
a e summa ized in Table 2.Figu e 6(c) illus a es ha o
all composi ions, he main h ee pa e ns o HAp appea
a 2θ= 26°, 32° and 46°, ha a e a ibu ed o (002), (211),
and (310) la ice planes in he HAp c ys als, which is also
aconfi ma ion o he bioac i i y o MBGNs and HAp
o ma ion.
33,48,68,74
Besides, i can be concluded, ha
e en hough FA appea ed o s abilize he films due o
addi ional c osslinking o GEL wi hin he fi s 7 days (as
men ioned abo e), i does no hinde he con ac o
MBGNs wi h SBF and he consequen o ma ion o a
HAp laye on he su ace om day 7, as i was o films
wi hou FA. The e o e, FA does no ha e a nega i e
impac on he bioac i i y o ADA-GEL-MBGN samples.
Thus, wi h ADA-GEL-FA-MBGN hyd ogels, cons uc s
wi h inc eased s abili y and bioac i e p ope ies we e
c ea ed.
Bide e al. 539

To addi ionally confi m he bioac i e p ope ies o he
MBGNs, pu e 0.1% (w/ ) and 0.5% (w/ ) MBGNs we e
imme sed in SBF solu ion o 7, 14 and 21 days o incu-
ba ion. The Si and Ca ion elease was measu ed wi h
ICP-OES ( esul s shown in supplemen a y pa in Figu e
S3). Howe e , a sligh ly lowe Si ion elease was measu ed
compa ed o Ca. This beha iou migh be explained due o
he ac ha Ca is loosely bonded in he silica e ne wo k.
Figu e 6. Confi ma ion o he o ma ion o HAp on he su ace o ADA-GEL-0.1%MBGN, ADA-GEL-0.5%MBGN, ADA-GEL-FA-0.1%
MBGN and ADA-GEL-0.5%MBGN samples a e being imme sed in SBF o 28 days wi h (a) SEM-EDS analysis (scale ba : 200 nm), (b)
ART-FTIR analysis and (c) XRD pa e ns.
540 Jou nal o Bioma e ials Applica ions 39(6)
Si ions a e eleased slowly and in a sus ained manne . Due o
he high su ace a ea o MBGNs, ini ially a as eac ion
occu ed a he in e ace which consequen ly lea es o he
p ecipi a ion o he calcium phospha e laye . This CaP laye
can also ac as a ba ie o he as e elease o Si ions.
Mo eo e , he lowe Si elease migh be explained due o he
supe sa u a ion o Si-ions in he solu ion. Due o he high
su ace a ea o he pa icles, ini ially a quick eac ion (ion
exchange) be ween SBF and MBGNs occu s which esul s
in o ma ion o he HAp laye . This HAp laye can es ic o
slow down he pa icle deg ada ion, which can be seen by
he low elease o Si ions.
Mechanical cha ac e iza ion
Comp ession es . One aim o his s udy was o de e mine he
capabili y o FA and MBGNs o inc ease he mechanical
s eng h o hyd ogels. Especially, due o he ac ha FA has
been epo ed o induce u he bonding wi h he polyme
ma ix, leading o an inc ease o mechanical s eng h o
hyd ogels.
75,76
Mo eo e , inco po a ion o MBGNs should
imp o e he mechanical p ope ies o hyd ogels.
33,77
Fo
his eason, he measu emen o he e ec i e modulus is
c ucial and was pe o med o all used hyd ogels (lis ed in
Table 1) in comp ession using a Mic o es e . Figu e 7(a)–(d)
illus a es he e ec i e modus o all composi ions. To en-
su e a be e compa ison o he composi ions among each
o he and o imp o e he isualiza ion o he FA and MBGN
impac on he e ec i e modulus, esul s a e p esen ed in
di e en g aphs. In Figu e 7(a) a clea end is isible ha
o ce ain ime poin s a highe e ec i e modulus was
measu ed o ADA-GEL-FA samples, which migh be
explained by he addi ional c osslinking o FA o GEL.
21
Howe e , he esul s a e no significan ly di e en . In
Figu e 7(b) ADA-GEL-0.1%MBGN and ADA-GEL-0.5%
MBGN samples a e compa ed using nea ADA-GEL
samples as e e ence ma e ial. A each ime poin an in-
c ease o he e ec i e modulus o MBGN con aining
samples is seen. This esul can be explained due o he Ca
ion elease om MBGNs, which leads o a mo e s abilized
ADA-GEL ma ix. In e es ingly, he composi ions wi h
lowe MBGN con en (0.1% (w/ )) seem o exhibi a highe
e ec i e modulus han ADA-GEL-0.5%MBGN samples.
This esul could confi m he assump ion ha agglome a es
o MBGNs a high concen a ions could dis up he in e nal
ma ix o ADA-GEL hyd ogel, leading o lowe mechanical
p ope ies. Simila beha iou is isible in Figu e 7(c) whe e
ADA-GEL-FA-MBGN samples we e compa ed using
ADA-GEL-FA as a e e ence ma e ial. In his compa ison
ADA-GEL-FA-0.1%MBGN samples show he highes e -
ec i e modulus measu ed du ing he en i e 28 days o
incuba ion compa ed o pu e ADA-GEL-FA samples,
which migh be again explained by he addi ional c oss-
linking wi h Ca ions eleased om MBGNs. The inc ease in
s i ness due o he p esence o ino ganic fille s was al eady
epo ed by Wei e al.
78
and Ma elli e al.,
79
which s udied
he mechanical p ope ies o collagen hyd ogels loaded wi h
nanosized BG pa icles incuba ed in SBF. They explained
hei inc ease o s i ness wi h he mine aliza ion beha iou
due o incuba ion in SBF solu ion. Howe e , since in ou
s udy films we e imme sed in cell cul u e medium he e-
sul s a e di ficul o compa e. On he o he hand, ADA-
GEL-FA-0.5%MBGN samples show a lowe e ec i e
modulus compa ed o ADA-GEL-FA-0.1%MBGN sam-
ples, which mos likely e e o he dis up ion o he en i e
hyd ogel ne wo k wi h highe pa icle concen a ion. A
simila esul was ound by Heid e al.
80
who in es iga ed
he mechanical p ope ies o ADA-GEL hyd ogels loaded
wi h 0.1% and 0.5% (w/ ) bioac i e ino ganic fille s (BIF).
Du ing he en i e incuba ion ime o 21 days, he modulus
alues anged be ween 3.7 and 6.5 kPa showing highe
moduli o samples wi h lowe 0.1% (w/ ) MBGN con en
compa ed o 0.5% (w/ ) MBGN loaded samples. They
explained his beha iou wi h an unp edic able loca ion o
pa icles be ween he co alen bonding o ADA and GEL
leading o a dis up ion o he Schi ’s base o ma ion and
hus a dec ease o in e nal s abili y o he ADA-GEL-based
samples which migh be an addi ional explana ion o he
p esen esul s and simila mechanical beha iou .
80
Com-
pa ing pu e ADA-GEL-MBGN samples wi h ADA-GEL-
FA-MBGN samples in Figu e 7(d) o all measu ed ime
poin s one can obse e an inc eased e ec i e modulus o
samples con aining FA compa ed o pu e MBGN samples.
Mo eo e , a dec ease o e ec i e modulus o 0.5% (w/ )
composi ions was measu ed indica ing he dis up ion o he
hyd ogel ne wo k due o high MBGN con en . Howe e ,
his beha iou becomes no significan ly di e en wi h
inc easing incuba ion ime. Ano he eason o he de-
c eased mechanical s abili y o ADA-GEL-based samples
wi h high 0.5% (w/ ) MBGN con en is likely ela ed o he
accele a ed deg ada ion men ioned abo e compa ed o
samples wi h 0.1% (w/ ) MBGNs. Howe e , i should be
men ioned ha o comp ession es s small films we e used
compa ed o he samples used o he deg ada ion s udy,
which migh a ec he deg ada ion beha iou and hus
explain he dec ease o mechanical s abili y o all com-
posi ions. The olume o su ace a io is highe o samples
used o comp ession es ing leading o a la ge su ace
exposed o he su ounding cell cul u e medium. Obse ing
Table 2. Summa y o FTIR peaks (see Figu e 6(b) and (c))
Vib a ion Wa enumbe (cm
1
) Re s
S e ching mode o PO
43-
1038, 1020
68,72
Vib a ion o HPO
42-
875, 959
68,72
Bending mode o CO
32-
ib a ion 875
72
Bending mode o PO
43-
ib a ion 560, 600
9,72,73
Bide e al. 541
Figu e 7. Compa ison o he e ec i e modulus o : (a) ADA-GEL and ADA-GEL-FA hyd ogel samples, (b) ADA-GEL-0.1%MBGN and
ADA-GEL-0.5%MBGN hyd ogel wi h ADA-GEL as e e ence and (c) ADA-GEL-FA-0.1%MBGN and ADA-GEL-FA-0.5%MBGN
hyd ogel wi h ADA-GEL-FA as e e ence. (d) ADA-GEL-0.1%MBGN, ADA-GEL-0.5%MBGN, ADA-GEL-FA-0.1%MBGN and ADA-
GEL-FA-0.5%MBGN hyd ogel. All samples we e incuba ed o e 28 days o incuba ion in cell cul u e medium. (e) Complex iscosi ies o
all composi ions using he equency sweep. ( ) Deg ee o c osslinking. S a is ical analysis was pe o med ia one-way ANOVA using
he Bon e oni es wi h p< 0.05 = *, p< 0.01 = ** and p< 0.001 = ***. n.s. = no significan di e en .
542 Jou nal o Bioma e ials Applica ions 39(6)
all composi ions, he e ec i e modulus o he hyd ogels
dec eases a e being imme sed in cell cul u e medium om
day 1, howe e , i main ained a ela i ely cons an me-
chanical s abili y o e he o al incuba ion pe iod o 28 days.
Especially ADA-GEL and ADA-GEL-FA samples do no
show a significan dec ease o mechanical p ope ies a e
imme sion, while he e ec i e modulus o he o he com-
posi ions (wi h MBGNs) d ops significan ly. Addi ionally,
his esul migh be explained wi h he hyd ophilic be-
ha iou o MBGNs, which can abso b wa e a e being
imme sed, hus leading o an accele a ed dec ease o me-
chanical p ope ies.
24
Dis le e al.
14
o ins ance in es i-
ga ed ADA-GEL hyd ogels c osslinked wi h CaCl
2
-
solu ions wi h a a ia ion o mTG concen a ions. Con-
side ing hei e ec i e modulus o samples c osslinked
wi h a 2.5% (w/ ) mTG solu ion, as i was done in ou s udy,
hey epo ed alues in a compa able ange o 5 –10 kPa.
Mo eo e , hey men ioned ha a s i ness o up o 120 kPa
migh be achie ed by inc easing he mTG concen a ion o
10% (w/ ).
14
Ou measu ed e ec i e moduli a e also
compa able wi h hose epo ed by Sa ke e al.,
81
who
measu ed a educed Young’s modulus o compa able
ADA-GEL hyd ogels (2.5%ADA and 2.5%GEL in 1:1 a-
io) o ∼10 kPa be o e incuba ion. E en hough an inc ease
o e ec i e modulus o he hyd ogels due o he p esence o
FA and MBGNs is measu ed in ou s udy, he mechanical
p ope ies o he p esen sca olds may no be enough o
hem o suppo loads in he con ex o bone egene a ion.
On he o he hand, he e ec i e modulus in comp ession
e ealed applica ion po en ial in con ac wi h so issues
such as kidney (5-10 kPa),
82
li e (1-6.5 kPa),
83
ca diac
muscle (up o 8 kPa),
84
o ca ilage (15-20 kPa).
85
De-
pending on he used cell ype he subs a e s i ness o cell
g ow h and p oli e a ion should ulfil ce ain equi emen s.
Due o he MBGN con en in ou samples, he ma e ial is in
p inciple in ended o bone TE applica ions. The e o e, o
he di ec cell s udy (see sec ion “Di ec es ”) and 3D bi-
op in ing s udy (see sec ion “3D biop in ing”) he p e-
os eoblas ic cell line MC3T3-E1 was used o in es iga e he
cell beha iou . As epo ed by Dis le e al.
14
, i is possible
o inc ease he mechanical s i ness by inc easing mTG and
CaCl
2
concen a ion which migh be conside ed o u u e
s udies.
Rheological e alua ion. The heological p ope ies o ADA-
GEL-based hyd ogels we e analysed o assess hei 3D
p in abili y. The s ain a e o ampli ude sweep was se a
10% based on a p e ious pa ame e iden ifica ion es .
20
The e o e, o he p esen s udy a cons an elonga ion os-
cilla ion s ain o 10% was used o measu e he equency
sweep es o FA and/o MBGN con aining ADA-GEL
hyd ogels o all used inks (lis ed in Table 1). In Figu e 7(e)
i is shown ha he complex iscosi y dec eases wi h in-
c easing angula equency o all used inks. This beha iou
is cha ac e is ic o he shea hinning beha iou o ma e-
ials.
86
This p ope y is c ucial since shea hinning allows
ma e ials o be injec ed using a shea o ce and hey egain
hei ini ial s uc u e a e emo al o ha o ce. This be-
ha iou allows hem o be ex uded o c ea e 3D p in ed
s uc u es.
87
A 1 ad/s he iscosi y is be ween 6 and
12 Pa∙s, whe eas he highes complex iscosi y was mea-
su ed o ADA-GEL-FA-0.1%MBGN inks and he lowes
o ADA-GEL-0.5%MBGN. Wi h inc easing angula e-
quencies o composi ions wi h he highes MBGN con en ,
ADA-GEL-0.5%MBGN and ADA-GEL-FA-0.5%MBGN,
he lowes iscosi ies (0.4 Pa∙s and 0.5 Pa∙s) we e mea-
su ed, espec i ely. This phenomenon migh be explained
by he ac ha a highe amoun o MBGNs could dis u b he
in e nal Schi ’s base o ma ion be ween ADA and GEL
leading o a lowe iscosi y o he hyd ogel. These esul s
co ela e wi h he obse a ions epo ed in sec ion “Deg ee
o c osslinking”, whe e a dec easing deg ee o c osslinking
wi h inc easing MBGN con en was shown. On he o he
hand, he addi ion o 0.1% (w/ ) MBGNs and FA seems o
s abilize he inks esul ing in he highes measu ed is-
cosi ies o ADA-GEL-FA and ADA-GEL-FA-0.1%
MBGN inks. This esul migh be explained by he addi-
ional in e nal c osslinking due o he eleased Ca ions om
MBGNs and he addi ional c osslinking be ween FA and
GEL.
21,55
The addi ional in e nal c osslinking is ad an a-
geous o ensu e a highe esolu ion o 3D p in ed sca olds.
In e es ingly, Zhu e al.
88
e alua ed he heological p op-
e ies o ADA-GEL hyd ogels loaded wi h bo h MBGNs
and amine- unc ionalized MBGNs (AMBGN). The unc-
ionaliza ion o MBGNs wi h NH
2
-g oups was im-
plemen ed o es ablish addi ional bonding wi h GEL chains.
This addi ional bonding o GEL is compa able wi h wha
was aimed a in his wo k wi h FA. In hei s udy, he
unc ionalized pa icles showed an inc eased iscosi y in
compa ison o he con ol ma e ial, howe e , a concen a-
ion o 1% (w/ ) MBGNs also led o a dec ease in iscosi y
and e ec i e modulus. Addi ionally, Heid e al.
80
also e-
po ed a dec ease in complex iscosi y wi h he inco po-
a ion o 0.5% (w/ ) bioac i e ino ganic fille s (BIFs)
compa ed o he composi ion wi h 0.1% (w/ ) BIFs.
80
A
dec ease o iscosi y and e ec i e modulus (see sec ion
“Comp ession es ”) wi h inc easing MBGN con en was
also confi med in ou s udy. Conside ing he heological
esul s, i migh be concluded ha he mos sui able hy-
d ogels o 3D p in ing a e ADA-GEL-FA and ADA-GEL-
FA-0.1%MBGN, which will be u he discussed in he nex
sec ion.
Assessmen o 3D p in abili y
The 3D p in abili y was assessed using di e en es s,
named FFT, FCT, and GST. This e alua ion aimed o
comp ehend he quali a i e and quan i a i e e ec s o FA
Bide e al. 543
and MBGNs on he esolu ion, shape fideli y, and cohesion
o ex uded 3D p in ed ADA-GEL sca olds. The ou comes
o hese es s we e u ilized o selec he mos app op ia e
inks. Subsequen ly, he op imal inks we e employed o a
mo e de ailed 3D p in ing assessmen .
FFT. In Figu e 8(a), he co ela ion be ween he used
segmen leng h (
s
) and filamen hickness (
)(
s
/
)is
plo ed agains he filamen dis ance (
d
). The pho os used
o e alua ing his es a e shown in Figu e 8(b). As he a io
(
s
/
) app oaches 1, he e is a highe p obabili y ha he
s ains will exhibi a squa e geome y along he edges while
main aining consis en hicknesses. This sugges s a s eady
p in ing o segmen s.
87
Howe e , i was no possible o
achie e a 0.5 mm filamen dis ance esolu ion o he p in ed
inks due o he me ging o s ains. Fo all inks, an inc ease in
filamen dis ance om 1.5 mm o 2 mm co esponds o a
highe esolu ion, esul ing in an
s
/
a io close o 1.
Con e sely, when dis ances dec ease om 1 mm o
0.75 mm, fluc ua ions indica e s ands using oge he ,
leading o an inc ease o he
s
/
a io. A small dis ances,
ADA-GEL-FA and ADA-GEL-FA-0.1%MBGN end o be
close o 1 compa ed o o he inks, indica ing hei sui -
abili y o 3D p in ing. This beha iou aligns wi h he
iscosi y measu emen s discussed in he sec ion “Rheo-
logical e alua ion”, showing he highes iscosi y o hese
wo inks and his may be he eason o he ob ained bes -
p in ed pa e n. Mo eo e , he enhanced p in abili y can be
explained by he addi ional c osslinking o FA and GEL.
21
The elease o Ca ions om MBGNs seems o lead o a mo e
s able p in ing, as epo ed elsewhe e.
80
Howe e , samples
wi h high MBGN con en in ADA-GEL-0.5%MBGN and
ADA-GEL-FA-0.5%MBGN show low- esolu ion p in ing,
indica ed by a highe
s
/
a io a small dis ances. This esul
migh be explained by he dec eased iscosi y o hese inks
men ioned abo e, and he assump ion ha 0.5% MBGN
con en migh dis up he hyd ogel ne wo k, leading o
inc eased usion o s ands du ing p in ing. Summa izing,
FFT indica ed he sui abili y o ADA-GEL-FA and ADA-
GEL-FA-0.1%MBGN inks o 3D p in ing.
FCT. The FCT is used o assess he maximum dis ance
be ween wo objec s, o e which a p in ed s and does no
collapse. This is pa icula ly c ucial o po ous s uc u es o
p e en he ea ing down o s ands o e po es. Fo he
e alua ion o he deflec ion angle, pho os o p in ed s ands
on op o he pilla s we e used, as shown in Figu e 8(d). The
ela ionship be ween he deflec ion angle and pilla dis-
ances is illus a ed in Figu e 8(c). I is e iden ha , ac oss
all p in ed composi ions, he deflec ion inc eases as he
dis ance be ween pilla s inc eases. Fo ADA-GEL-0.5%
MBGN, e en a a small gap dis ance o 2 mm, a deflec ion
angle o 4 ± 1° could be measu ed, possibly due o he
inc eased weigh o inks a ibu ed o he highe con en o
MBGNs esul ing in a as collapse o s ands. Especially
wi h inc easing gap dis ances, his phenomenon is mo e
isible o ADA-GEL-0.5%MBGN and ADA-GEL-FA-
0.5%MBGN inks, eaching alues o 11 ± 1° and 26 ± 2°,
espec i ely. This is ollowed by ADA-GEL and ADA-
GEL-0.1%MBGN inks. Howe e , ADA-GEL-0.1%MBGN
shows lowe alues compa ed o pu e ADA-GEL inks,
indica ing he beneficial addi ion o low MBGN con en due
o he inne c osslinking by Ca ions eleased by MBGNs.
80
O e all, ADA-GEL-FA and ADA-GEL-FA-0.1%MBGN
inks show he smalles deflec ion angle wi h inc easing gap
dis ances, highligh ing hem as he mos sui able inks. The
imp o ed p in abili y o hese wo inks can be a ibu ed o
he addi ional c osslinking o FA and GEL
21
and he
p esence o Ca ions eleased by MBGNs
80
men ioned
abo e. Mo eo e , he inc eased iscosi y and inc eased
deg ee o c osslinking (see sec ion “Deg ee o c oss-
linking”) o hese wo inks can also con ibu e o he im-
p o ed p in ing p ope ies compa ed o o he inks.
GST. A e assessing he bes esolu ion o inks (FFT) and
he abili y o c ea e po ous s uc u es (FCT), 3D p in ed
g ids we e gene a ed in e ms o a GST o a quan i a i e
e alua ion o shape fideli y and geome y accu acy in 3D
p in ing. The diagonal c ossing a io (DCR) se ed as an
indica o o assess he po es o 3D p in ed sca olds, de-
e mining whe he he s ands main ained hei ideal po e
shape o used when s acked.
43
Hazu e al.
43
conside ed
354 µm as an ideal diagonal while p in ing 1 × 1 cm
2
g ids
wi h s ands o 250 µm. In his s udy, he ideal diagonal
(d
ideal
) o a po e was calcula ed by measu ing he po e wid h
and heigh (see Figu e 3(c),w
,x
,w
h,x
, x = 5 eplica es). The
measu emen was epea ed fi e imes wi hin one ligh
mic oscope image o a 3D p in ed sca old. The ligh mi-
c oscopy images used o he e alua ion o po es and s u s
a e displayed in Figu e 8(e). Acco ding o equa ion (4),
d
ideal
was calcula ed. The ac ual diagonal (d
m
) o he
e alua ed po es was also measu ed wi h ImageJ. Finally, he
DCR was de e mined acco ding o equa ion (5) as he
quo ien be ween d
m
and d
ideal
, as illus a ed in Figu e 8( ).
The ideal DCR alue was 1.0, wi h a dec ease in alue
owa ds ze o indica ing a ounde po e. I is obse ed ha
ADA-GEL-FA, ADA-GEL-0.1%MBGN, and ADA-GEL-
FA-0.1%MBGN inks exhibi ed he closes alues, while
samples con aining 0.5% (w/ ) MBGNs led o a dec eased
alue esul ing in s and usion. The s abiliza ion o po es in
he o me inks may be a ibu ed o he addi ional c oss-
linking o FA and GEL
21
and he elease o Ca ions om
MBGNs wi hin he inks.
80
Howe e , an excessi ely high
con en o MBGNs leads o he opposi e e ec . Simila
imp o emen s in 3D p in ing shape fideli y we e confi med
by he ela ed es s (FFT and FCT) discussed abo e. Fo he
GST, a nozzle wi h a diame e o 250 µm was used, con-
side ing he ideal s u size as 250 µm. To e alua e he s u s,
544 Jou nal o Bioma e ials Applica ions 39(6)

Figu e 8. (a) FFT wi h fi e di e en s u dis ances, anging om 2 mm o 0.5 mm. (b) Pho os o s uc u es p in ed o FFT. (c)
Deflec ion angle o he FCT es s gap dis ance. (d) Pho os o he s ands. Scale ba : 1 cm. (e) Ligh mic oscopy images o all p in ed
ADA-GEL-based inks. Scale ba : 2 mm. ( ) DCR and (g) s u wid h o all composi ions. (h) Angula de ia ion o acu e and ob use angles
o ADA-GEL-FA and ADA-GEL-FA-0.1%MBGN. (i) Ligh mic oscopy images o s a -shaped s uc u es. Scale ba : 1 mm and 2 mm.
S a is ical analysis was pe o med ia one-way ANOVA using he Bon e oni es wi h p< 0.05 = *, p< 0.01 = ** and p< 0.001 = ***.
Bide e al. 545
fi e di e en s u wid hs e ically and ho izon ally (see
Figu e 3(c),s
,x
,s
h,x
, x = 5 eplica es) on one ligh mi-
c oscopy pic u e we e measu ed. In Figu e 8(g), he mean o
he measu ed s u s is shown, wi h he ideal s u o 250 µm
wid h ma ked wi h a ed line. 3D sca olds p in ed wi h
ADA-GEL-FA, ADA-GEL-0.1%MBGN, and ADA-GEL-
FA-0.1%MBGN inks could be p in ed wi h s u s close o
250 µm compa ed o he o he inks. This beha iou co ela ed
wi h esul s es ablished o he DCR analysis, again con-
fi ming ADA-GEL-FA and ADA-GEL-FA-0.1%MBGNs as
he mos sui able (p in able) inks.
Complex s a -shape p in ing. In FFT, FCT, and GST, i was
indica ed ha ADA-GEL-FA and ADA-GEL-FA-0.1%
MBGN inks appea ed o be he mos sui able o 3D
p in ing. To es his finding, hese wo inks we e selec ed
o ab ica ing mo e complex and in ica e s uc u es. S a
shapes we e p in ed o quali a i ely and quan i a i ely
assess he ep oducibili y o edges and e ices.
Figu e 8(i) shows mic oscopy images o he en i e s a
(uppe images, scale ba : 2 mm), o poin s o he s a
(lowe images, scale ba : 1 mm), and o he in e sec ions
be ween he poin s (middle images, scale ba : 1 mm)
p in ed wi h ADA-GEL-FA and ADA-GEL-FA-0.1%
MBGN. In Figu e 8(h), he esul s o angle de ia ion in
pe cen age, calcula ed acco ding o equa ion (6), a e
displayed. The expe imen al acu e and ob use angle (θe)
was measu ed by connec ing he e ex o he in e sec-
ion, as illus a ed in Figu e 3(d). The esul s depic ed in
Figu e 8(h) e eal ha he ADA-GEL-FA p in ed
s uc u es exhibi s a highe de ia ion om he heo e ical
angle compa ed o ADA-GEL-FA-0.1%MBGN ink. This
end unde lines he posi i e impac o Ca ions elease
in o he polyme ne wo k.
80
Howe e , he di e ence is
no significan . Conside ing he posi i e end es ablished
in his p in ing expe imen , ADA-GEL-FA-0.1%MBGN ink
wasused op in la ge g idso 1.5×1.5cm
2
wi h 1, 4, and
8 laye ed sca olds. A bone shape s uc u e was also p in ed.
Resul s a e displayed in Figu e S4 (see supplemen a y pa ).
Success ul p in ing o a bone sca old (Figu e S4(a))and
success ul p in ing up o 8 laye s (Figu e S4(b))canbeob-
se ed. Figu e S4(c) addi ionally shows SEM images o a
4-laye sca old in 3 di e en magnifica ions. Mo eo e , ex-
cellen handling o sca olds was confi med a e c osslinking
he samples wi h a c osslinking solu ion composed o 0.1 M
CaCl
2
and 2.5% (w/ ) mTG.
In conclusion, he inco po a ion o FA and 0.1%MBGNs
in o ADA-GEL inks leads o an inc eased iscosi y a -
ibu ed o Ca ions eleased by MBGNs
80
and he addi ional
c osslinking o GEL and FA.
21
Fu he mo e, he ising
deg ee o c osslinking and, consequen ly, an inc eased
o ma ion o Schi ’s base, could con ibu e o enhanced
p in abili y.
In i o cy ocompa ibili y assessmen
Di ec es . In o de o assess whe he he ma e ials p o ide
a sui able en i onmen o cells, MC3T3-E1 cells we e
di ec ly inco po a ed in o all hyd ogels (composi ions lis ed
in Table 1) o 24 h and 7 days o incuba ion. A e ce ain
ime poin s he cell ac i i y was analysed by a WST-8 assay
o 4 h. Subsequen ly, he cell iabili y was in es iga ed by a
Calcein AM and DAPI s aining. The medium du ing in-
cuba ion ime was changed wice a week. Figu e 9(b) shows
he WST-8 esul s (op ical densi ies a 450 nm). A e
24 h o incuba ion, MC3T3-E1 cells showed he lowes cell
ac i i y o 0.05 wi hin ADA-GEL samples, while hey
eached he highes cell ac i i y o 0.12 when inco po a ed
in ADA-GEL-FA-0.1%MBGN samples. A e 7 days o
incuba ion an inc ease o ac i i y is isible o all samples,
anging om 0.1 o ADA-GEL up o 0.18 o ADA-GEL-
FA-0.1%MBGN samples. Zehnde e al.
89
epo ed he cell
ac i i y o he MG-63 (os eoblas -like) cell line inco po a ed
in o ADA-GEL samples, which was a ound 0.05. Howe e ,
2.5%ADA and 2.5%GEL (w/ ) concen a ions we e used in
ha s udy, which migh explain he highe cell ac i i y
epo ed in ou s udy in which a highe GEL concen a ion
(3.75% (w/ )) was used. The p esence o a highe GEL
concen a ion p o ides mo e RGD sequences o cell a -
achmen likely esul ing in highe cell ac i i ies. The in-
c ease o cell ac i i y wi h inc easing incuba ion ime migh
also be explained wi h he g adual deg ada ion o he hy-
d ogels du ing he fi s week, as discussed in sec ion “In
i o deg ada ion/swelling beha iou ”). The deg ada ion
leads o highe hyd ogel ne wo k po osi y esul ing in be e
cell mig a ion and p oli e a ion inside he hyd ogels.
53
Since he lowes ac i i y was measu ed o ADA-GEL
samples, he addi ion o FA and MBGNs appea ed o be
beneficial o MC3T3-E1 cells. The posi i e e ec o FA on
cell beha iou has been al eady epo ed in p e ious
s udies.
90,91
The inc ease o cell ac i i y due o he p esence
o MBGNs was also confi med o MC3T3-E1 cells by
Mona a i e al.
33
and o mouse de mal fib oblas cells by
Weie al.
78
Mo eo e , a posi i e e ec o MBGNs on bone
cell ac i i y has been epo ed by Wu e al.,
92
who s udied
MG-63 cells encapsuled in a MBGN/sodium algina e/
gela in hyd ogel.
92
I is no able ha highe cell ac i i ies
a e epo ed o samples wi h lowe 0.1% (w/ ) MBGN
con en compa ed o samples wi h highe 0.5% (w/ )
MBGN concen a ion o bo h measu ed ime poin s. This
esul migh be explained by he apid ion elease, which
migh inc ease he pH o he local en i onmen .
93
Com-
pa able esul s we e epo ed by Ye e al.
94
when examining
he ac i i y o bone ma ow-de i ed mesenchymal s em
cells (BMSCs) wi hin an algina e and gela in hyd ogel
loaded wi h MBGNs a a ying concen a ions. Specifically,
hey obse ed a decline in cell ac i i y a concen a ions
exceeding 10% (w/ ) a e 7 days o incuba ion, a ibu ed
546 Jou nal o Bioma e ials Applica ions 39(6)
Figu e 9. (a) MC3T3-E1 cell iabili y du ing di ec cell s udy and cell ac i i ies o embedded cells in o all bioinks a e (b) di ec cell s udy
and (d) 3D biop in ing wi hin 24 h and 7 days o incuba ion. Fluo escence mic oscopy images o he cells a e (c) di ec cell s udy and
(e) 3D biop in ing, including ADA-GEL as a con ol. G een: Calcein AM, blue: DAPI. Scale ba 200 μm and 500 µm. S a is ical analysis
was pe o med ia one-way ANOVA using he Bon e oni es wi h p< 0.05 = *, p< 0.01 = ** and p< 0.001 = ***.
Bide e al. 547
o he alkaline en i onmen c ea ed by he MBGNs.
Howe e , his di e ence was no e iden a e 21 days,
sugges ing a long- e m benefi o he p esence o MBGNs in
cell cul u es.
94
The highes ac i i y o MC3T3-E1 cells
wi hin ADA-GEL-FA-0.1%MBGN samples indica ed he
mos p omising composi ion owing o i s a ou able
combina ion o FA and a low concen a ion o 0.1% (w/ )
MBGNs. Following he WST-8 assay a Calcein AM and
DAPI s aining was pe o med and isualized by aking
fluo escence images, as illus a ed in Figu e 9(c) a e 24 h
and 7 days o incuba ion. In all images a high cell dis i-
bu ion and high cell densi y can be obse ed. In Figu e 9(a)
he iabili y o MC3T3-E1 cells is shown, which was
calcula ed by conside ing he a io be ween ali e cells
(Calcein AM s aining, g een do s in Figu e 9(c)) and he
o al amoun o cells (DAPI s aining, blue do s in
Figu e 9(c)). Fo all samples a high MC3T3-E1 cell iabili y
wi hin all hyd ogel composi ions was de e mined. A e
7 days o incuba ion a sligh ly inc ease o cell iabili y o
ADA-GEL-FA and ADA-GEL-FA-0.1%MBGN samples
(95% and 96%, espec i ely) compa ed o nea ADA-GEL
samples (91%) can be seen bu hese alues a e no sig-
nifican ly di e en . I can be concluded ha ADA-GEL-FA
and ADA-GEL-FA-0.1%MBGN hyd ogels a e he mos
p omising inks o MC3T3-E1 cells a e 7 days o
incuba ion.
3D biop in ing. To assess he impac o shea s ess du ing
3D p in ing on MC3T3-E1 cells, a 3D biop in ing es was
pe o med. The MC3T3-E1 cell ac i i y wi hin 3D p in ed
hyd ogel sca olds was measu ed wi h a WST-8 assay a e
24 h and 7 days o incuba ion. In Figu e 9(d) he op ical
densi ies measu ed a 450 nm a e displayed, whe eas in
Figu e 9(e) fluo escence mic oscopy images o inco po a ed
MC3T3-E1 cells wi hin 3D p in ed sca olds o all com-
posi ions (Table 1) a e shown. In all fluo escence mic os-
copy images, a homogeneous cell dis ibu ion and high cell
densi y can be obse ed. In Figu e 9(d) o all samples an
inc ease in cell ac i i y wi h inc easing incuba ion ime was
measu ed. A e 7 days o incuba ion, he highes cell ac-
i i ies o 3D p in ed MC3T3-E1 cells wi hin hyd ogels
we e measu ed o ADA-GEL-FA and ADA-GEL-FA-0.1%
MBGN sca olds wi h 0.25 ± 0.01 and 0.24 ± 0.02, e-
spec i ely. Fo bioinks wi hou FA, ADA-GEL, ADA-GEL-
0.1%MBGN and ADA-GEL-0.5%MBGN, lowe cell ac-
i a es we e measu ed wi h 0.18 ± 0.05, 0.21 ± 0.04 and
0.20 ± 0.03, espec i ely, indica ing he posi i e e ec o FA
a e 7 days o incuba ion. The inc ease o cell ac i i y due
o he p esence o FA and MBGNs was al eady confi med
du ing he di ec cell s udy (see abo e). Especially based on
he an i-inflamma o y p ope ies o FA
95
and he cell
s imula ing p ope ies o MBGNs
96–98
he high cell ac i -
i ies can be explained. Mo eo e , he inc ease o cell ac-
i i ies wi h inc easing incuba ion ime can be explained by
he deg ada ion o he 3D p in ed sca olds (discussed in
sec ion “In i o deg ada ion/swelling beha iou ”) esul ing
in an accele a ed con ac o WST-8 solu ion and hus im-
p o ed me abolism o MC3T3-E1 cells leading o highe
measu ed op ical densi ies. Mo eo e , deg ada ion c ea es a
highe po osi y wi hin he hyd ogel ne wo k ha enables a
acile infil a ion o MC3T3-E1 cells.
53
Zhu e al.
88
in-
co po a ed MG-63 and ST2 cells in o ADA-GEL-based
bioinks in o de o in es iga e he cell ac i i ies a e 3D
biop in ing. Thei esul s showed op ical densi ies be ween
0.4 and 0.7 o MG-63 and 0.15 –0.2 o ST2 cells a e
7 days o incuba ion, which a e compa able o he alues
measu ed o MC3T3-E1 cells in ou 3D biop in ing s udy.
Zehnde e al.
89
e alua ed he cellula ac i i y o MG-63
cells wi hin ADA-GEL sca olds o e 28 days o incuba-
ion. They measu ed an op ical densi y o a ound 0.2 a e
14 days o incuba ion.
89
In ou s udy, compa able ac i i ies
we e eached al eady a e 7 days o incuba ion indica ing
he beneficial e ec o he addi ion o FA
91
and MBGNs.
33
Mona a i e al.
33
seeded MC3T3-E1 cells on op o 3D
p in ed ADA-GEL-based cons uc s composed o MBGNs
and ica iin. A e a o al incuba ion ime o 6 days, he
au ho s epo ed he highes iabili y a e 24 h compa ed o
4 and 6 days o incuba ion. They explained hei dec ease in
cell ac i i y wi h he as ica iin elease, ha u ned ou o be
cy o oxic o cells.
33
Howe e , in ou s udy cells we e
di ec ly inco po a ed in o he inks and no seeded on op o
3D p in ed sca olds, hus cell ac i i ies a e di ficul o
compa e. In ou case he cell ac i i y inc eases wi h in-
c easing incuba ion ime, which indica es he beneficial
en i onmen o cells p o ided by FA and MBGNs.
The e o e, i can be concluded ha he addi ion o FA and
MBGNs is a p omising app oach o achie e a sui able
en i onmen o cell p oli e a ion wi hin 3D biop in ed
ADA-GEL sca olds. Besides ha , he esul s confi m a
sui able choice and adjus men o he p in ing pa ame e s,
which ensu e a good shape fideli y o 3D p in ed sca olds
wi hou ha ming he cells du ing he p in ing p ocess.
Vascula endo helial g ow h ac o -A (VEGF-A)
elease
The VEGF-A is a c ucial biomolecule in he angiogenesis
p ocess
88
and is sec e ed om ce ain cells including bone
ma ow s omal cells, fib oblas s and os eoblas s.
99
In
pa icula , VEGF-A is p oduced by cells ha s imula e he
o ma ion o new blood essels.
88
As epo ed by Zhu
e al.
88
he ela ionship be ween ascula iza ion and new
bone o ma ion is e y close in bone TE. The induc ion o
ascula iza ion is an impo an signal o success ul bone
egene a ion.
100
The e o e, in es iga ing whe he FA o
MBGNs wi hin ADA-GEL-based hyd ogels can enhance
angiogenesis and p o ide a be e en i onmen o bone
548 Jou nal o Bioma e ials Applica ions 39(6)
71. Gineb a MP, Fe n´
andez E, D iessens FCM, e al. Modeling
o he hyd olysis o α- icalcium phospha e. J Am Ce am Soc
1999; 82: 2808–2812. DOI: 10.1111/j.1151-2916.1999.
b02160.x.
72. Roguska A, Pisa ek M, And zejczuk M, e al. Su ace cha -
ac e iza ion o Ca-P/Ag/TiO2 nano ube composi e laye s on Ti
in ended o biomedical applica ions. JBiomedMa e Res
2012; 100: 1954–1962. DOI: 10.1002/jbm.a.34044.
73. Zheng K, Solodo nyk A, Li W, e al. Aging ime and
empe a u e e ec s on he s uc u e and bioac i i y o gel-
de i ed 45S5 glass-ce amics. J Am Ce am Soc 2015; 98:
30–38. DOI: 10.1111/jace.13258.
74. Bui XVand Dang TH. Bioac i e glass 58S p epa ed using an
inno a ion sol-gel p ocess. P ocess. Appl. Ce am 2019; 13:
98–103. DOI: 10.2298/PAC1901098B.
75. Bisca a J, Cha me e C, Masquelez N, e al. C osslinking o
gela in memb anes wi h e ulic acid o glu a aldehyde: e-
la ionship be ween gas pe meabili y and ena u a ion le el
o gela in iple helices. J Polym Sci B Polym Phys 2015; 53:
280–287. DOI: 10.1002/polb.23628.
76. Sche e S, Oes M and Rohn S. In e ac ions be ween phe-
nolic acids, p o eins, and ca bohyd a es—influence on
dough and b ead p ope ies. Foods 2021; 10: 2798. DOI: 10.
3390/ oods10112798.
77. Ba e o MEV, Medei os RP, Shea e A, e al. Gela in and
bioac i e glass composi es o issue enginee ing: a e iew.
J Func Bioma e 2023; 14: 23. DOI: 10.3390/j b14010023.
78. Wei L, Li Z, Li J, e al. An app oach o mechanical p ope y
op imiza ion o cell-laden algina e–gela in composi e bioink
wi h bioac i e glass nanopa icles. J Ma e Sci Ma e Med
2020; 31: 103–112. DOI: 10.1007/s10856-020-06440-3.
79. Ma elli B, Ghezzi CE, Mohn D, e al. Accele a ed mine -
aliza ion o dense collagen-nano bioac i e glass hyb id gels
inc eases sca old s i ness and egula es os eoblas ic
unc ion. Bioma e ials 2011; 32: 8915. DOI: 10.1016/j.
bioma e ials.2011.08.016.
80. Heid S, Becke K, Byun J, e al. Biop in ing wi h bioac i e
algina e dialdehyde-gela in (ADA-GEL) composi e bioinks:
ime-dependen in-si u c osslinking ia addi ion o calcium-
silica e pa icles unes in i o s abili y o 3D biop in ed
cons uc s. Biop in ing 2022; 26: e00200. DOI: 10.1016/j.
bp in .2022.e00200.
81. Sa ke B, Zehnde T, Ra h SN, e al. Oxidized algina e-
gela in hyd ogel: a a o able ma ix o g ow h and os eo-
genic di e en ia ion o adipose-de i ed s em cells in 3D.
ACS Bioma e Sci Eng 2017; 3: 1730–1737. DOI: 10.1021/
acsbioma e ials.7b00188.
82. Hando AM, Zhou Y, Halanski MA, e al. Tissue s i ness
dic a es de elopmen , homeos asis, and disease p og ession.
O ganogenesis 2015; 11: 1–15. DOI: 10.1080/15476278.
2015.1019687.
83. Chen F, Tian M, Zhang D, e al. P epa a ion and cha ac-
e iza ion o oxidized algina e co alen ly c oss-linked gal-
ac osyla ed chi osan sca old o li e issue enginee ing.
Ma e Sci Eng C 2012; 32: 310–320. DOI: 10.1016/j.msec.
2011.10.034.
84. Baniasadi H, Mashayekhan S, Fadaoddini S, e al. Design,
ab ica ion and cha ac e iza ion o oxidized algina e-gela in
hyd ogels o muscle issue enginee ing applica ions.
J Bioma e Appl 2016; 31: 152–161. DOI: 10.1177/
0885328216634057.
85. K elle T, Dis le T, Heid S, e al. Physico-chemical mod-
ifica ion o gela ine o he imp o emen o 3D p in abili y
o oxidized algina e-gela ine hyd ogels owa ds ca ilage
issue enginee ing. Ma e Des 2021; 208: 109877. DOI: 10.
1016/j.ma des.2021.109877.
86. Cuomo F, Co elice M and Lopez F. Rheological cha ac-
e iza ion o hyd ogels om algina e-based nanodispe sion.
Polyme s 2019; 11: 259. DOI: 10.3390/polym11020259.
87. Ashammakhi N, Ahadian S, Xu C, e al. Bioinks and bio-
p in ing echnologies o make he e ogeneous and biomi-
me ic issue cons uc s. Ma e Today Bio 2019; 1: 100008.
DOI: 10.1016/j.m bio.2019.100008.
88. Zhu H, Mona a i M, Zheng K, e al. 3D biop in ing o
mul i unc ional dynamic nanocomposi e bioinks inco po-
a ing Cu-doped mesopo ous bioac i e glass nanopa icles
o bone issue enginee ing. Small 2022; 18: e2104996.
DOI: 10.1002/smll.202104996.
89. Zehnde T, Sa ke B, Boccaccini AR, e al. E alua ion o an
algina e-gela ine c osslinked hyd ogel o bioplo ing. Bi-
o ab ica ion 2015; 7: 025001. DOI: 10.1088/1758-5090/7/
2/025001.
90. Doss HM, Sama pi a S, Ganesan R, e al. Fe ulic acid, a
die a y polyphenol supp esses os eoclas di e en ia ion and
bone e osion ia he inhibi ion o RANKL dependen NF-κB
signalling pa hway. Li e Sci 2018; 207: 284–295. DOI: 10.
1016/j.l s.2018.06.013.
91. Akh a MA, Ma io i CE, Con i B, e al. Elec opho e ic
deposi ion o e ulic acid loaded bioac i e glass/chi osan as
an ibac e ial and bioac i e composi e coa ings. Su Coa
Technol 2021; 405: 126657. DOI: 10.1016/j.su coa .2020.
126657.
92. Wu J, Miao G, Zheng Z, e al. 3D p in ing mesopo ous
bioac i e glass/sodium algina e/gela in sus ained elease
sca olds o bone epai . J Bioma e Appl 2019; 33:
755–765. DOI: 10.1177/0885328218810269.
93. Aguila -Rabiela AE, Leal-Egaña A, Nawaz Q, e al. In e-
g a ion o mesopo ous bioac i e glass nanopa icles and
cu cumin in o PHBV mic osphe es as biocompa ible
composi e o d ug deli e y applica ions. Molecules 2021;
26: 3177. DOI: 10.3390/molecules26113177.
94. Ye Q, Zhang Y, Dai K, e al. Th ee dimensional p in ed
bioglass/gela in/algina e composi e sca olds wi h p omo ed
mechanical s eng h, biomine aliza ion, cell esponses and
os eogenesis. J Ma e Sci Ma e Med 2020; 31: 77. DOI: 10.
1007/s10856-020-06413-6.
95. Ambo hi K, P asad NR and Balupillai A. Fe ulic acid in-
hibi s UVB- adia ion induced pho oca cinogenesis h ough
Bide e al. 555

modula ing inflamma o y and apop o ic signaling in Swiss
albino mice. Food Chem Toxicol 2015; 82: 72–78. DOI: 10.
1016/j. c .2015.04.031.
96. Zheng K, Kapp M and Boccaccini AR. P o ein in e ac ions
wi h bioac i e glass su aces: a e iew. Appl Ma e Today
2019; 15: 350–371. DOI: 10.1016/j.apm .2019.02.003.
97. Se gi R, Bellucci D and Cannillo V. A e iew o bioac i e
glass/na u al polyme composi es: s a e o he a . Ma e ials
2020; 13: 1–38. DOI: 10.3390/ma13235560.
98. Go us o ich AA, Roe he JA and Boccaccini AR. E ec o
bioac i e glasses on angiogenesis: a e iew o in i o and
in i o e idences. Tissue Eng Pa B 2010; 16: 199–207.
DOI: 10.1089/ en. eb.2009.0416.
99. Day RM. Bioac i e glass s imula es he sec e ion o an-
giogenic g ow h ac o s and angiogenesis in i o. Tissue
Eng 2005; 11: 768–777. DOI: 10.1089/ en.2005.11.768.
100. Bellucci D, B accini S, Chiellini F, e al. Bioac i e glasses and
glass-ce amics e sus hyd oxyapa i e: compa ison o angio-
genic po en ial and biological esponsi eness. JBiomedMa e
Res 2019; 107: 2601–2609. DOI: 10.1002/jbm.a.36766.
101. Chai Q, Zhang B, Da Y, e al. Enhancemen and epai o
degene a i e in e e eb al disc in a s using pla ele - ich
plasma/ e ulic acid hyd ogel. Ca ilage 2023; 14: 506–515.
DOI: 10.1177/19476035231157341.
102. Lin CM, Chiu JH, Wu IH, e al. Fe ulic acid augmen s an-
giogenesis ia VEGF, PDGF and HIF-1 alpha. JNu Biochem
2010; 21: 627–633. DOI: 10.1016/j.jnu bio.2009.04.001.
103. Li Z, Wang S, Li W, e al. Fe ulic acid imp o es unc ional
eco e y a e acu e spinal co d inju y in a s by inducing
hypoxia o inhibi mic oRNA-590 and ele a e ascula
endo helial g ow h ac o exp essions. F on Mol Neu osci
2017; 10: 183. DOI: 10.3389/ nmol.2017.00183.
104. Wang H, Zhang Y, Xia T, e al. Syne gis ic p omo ion o
blood essel egene a ion by as agaloside IV and e ulic
acid om elec ospun fib ous ma s. Mol Pha m 2013; 10:
2394–2403. DOI: 10.1021/mp400031y.
105. Zhang Y, Hu G, Li S, e al. P o-angiogenic ac i i y o as-
agaloside IV in HUVECs in i o and zeb afish in i o. Mol
Med Rep 2012; 5: 805–811. DOI: 10.3892/mm .2011.716.
106. Dashnyam K, El-Fiqi A, Bui ago JO, e al. A mini e iew
ocused on he p oangiogenic ole o silica e ions eleased
om silicon-con aining bioma e ials. J Tissue Eng 2017; 8:
2041731417707339. DOI: 10.1177/2041731417707339.
107. Kong CH, S e fiC, Shi Z, e al. De elopmen o mesopo ous
bioac i e glass nanopa icles and i s use in bone issue
enginee ing. J Biomed Ma e Res B Appl Bioma e 2018;
106: 2878–2887. DOI: 10.1002/jbm.b.34143.
108. Yang GW, Jiang JS and Lu WQ. Fe ulic acid exe s an i-
angiogenic and an i- umo ac i i y by a ge ing fib oblas
g ow h ac o ecep o 1-media ed angiogenesis. In J Mol
Sci 2015; 16: 24011–24031. DOI: 10.3390/ijms161024011.
109. Zhou Y, Shi M, Jones JR, e al. S a egies o di ec as-
cula isa ion using mesopo ous bioac i e glass-based bio-
ma e ials o bone egene a ion. In Ma e Re 2017; 62:
392–414. DOI: 10.1080/09506608.2016.1266744.
110. Hsu FY, Lu MR, Weng RC, e al. Hie a chically biomime ic
sca old o a collagen-mesopo ous bioac i e glass nanofibe
composi e o bone issue enginee ing. Biomed Ma e 2015;
10: 025007. DOI: 10.1088/1748-6041/10/2/025007.
111. Jung GY, Pa k YJ and Han JS. E ec s o HA eleased calcium
ion on os eoblas di e en ia ion. J Ma e Sci Ma e Med 2010;
21: 1649–1654. DOI: 10.1007/s10856-010-4011-y.
112. Re fi DM, Ogs on N, Jugdaohsingh R, e al. O hosilicic
acid s imula es collagen ype 1 syn hesis and os eoblas ic
di e en ia ion in human os eoblas -like cells in i o.
J. Bone Mine . Res. Res 2003; 32: 127–135. DOI: 10.1016/
S8756-3282(02)00950-X.
113. Kee ing PE, Ou sle MJ, Wiegand KE, e al. Zeoli e A
inc eases p oli e a ion, di e en ia ion, and ans o ming
g ow h ac o be a p oduc ion in no mal adul human
os eoblas -like cells in i o. J Bone Mine Res 1992; 7:
1281–1289. DOI: 10.1002/jbm .5650071107.
114. Zhou W, Chen B, Shang J, e al. Fe ulic acid a enua es
os eopo osis induced by glucoco icoid h ough egula ing
he GSK-3β/L p-5/ERK signalling pa hways. Phys In 2021;
108: 317–341. DOI: 10.1556/2060.2021.00180.
115. Du K, Li Z, Fang X, e al. Fe ulic acid p omo es os eogenesis
o bone ma ow-de i ed mesenchymal s em cells by in-
hibi ing mic oRNA-340 o induce β-ca enin exp ession
h ough hypoxia. Eu J Cell Biol 2017; 96: 496–503. DOI:
10.1016/j.ejcb.2017.07.002.
116. Halling Linde C, Ek-Rylande B, K umpel M, e al. Bone
alkaline phospha ase and a a e- esis an acid phospha ase:
po en ial co- egula o s o bone mine aliza ion. Calci Tissue
In 2017; 101: 92–101. DOI: 10.1007/s00223-017-0259-2.
117. Gho bani F, Kim M, Mona a i M, e al. Mussel-inspi ed
polydopamine deco a ed algina e dialdehyde-gela in 3D
p in ed sca olds o bone issue enginee ing applica ion.
F on Bioeng Bio echnol 2022; 10: 940070. DOI: 10.3389/
bioe.2022.940070.
556 Jou nal o Bioma e ials Applica ions 39(6)