Ci a ion: Moazeni, N.; Hesa aki, S.;
Behnamghade , A.; Esmaeilzadeh, J.;
O i e, G.; Dola shahi-Pi ouz, A.;
Bo han, S. Design and Manu ac u e
o Bone Cemen s Based on Calcium
Sul a e Hemihyd a e and Mg,
S -Doped Bioac i e Glass.
Biomedicines 2023,11, 2833.
h ps://doi.o g/10.3390/
biomedicines11102833
Academic Edi o : Vi iana
Di Giacomo
Recei ed: 11 Sep embe 2023
Re ised: 4 Oc obe 2023
Accep ed: 16 Oc obe 2023
Published: 18 Oc obe 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
biomedicines
A icle
Design and Manu ac u e o Bone Cemen s Based on Calcium
Sul a e Hemihyd a e and Mg, S -Doped Bioac i e Glass
Nazanin Moazeni 1, Saeed Hesa aki 1,*, Aliasgha Behnamghade 1, Ja ad Esmaeilzadeh 2, Go ka O i e 3,4,
Ali eza Dola shahi-Pi ouz 5and Shokou eh Bo han 6
1Nano echnology and Ad anced Ma e ials Depa men , Ma e ials and Ene gy Resea ch Cen e ,
Ka aj 31779-83634, Albo z, I an; [email p o ec ed] (N.M.); [email p o ec ed] (A.B.)
2Depa men o Ma e ials and Chemical Enginee ing, Es a ayen Uni e si y o Technology,
Es a ayen 96619-98195, No h Kho asan, I an; [email p o ec ed]
3NanoBioCel Resea ch G oup, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU),
01006 Vi o ia-Gas eiz, Spain; [email p o ec ed]
4Biomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN),
Ins i u e o Heal h Ca los III, 28029 Mad id, Spain
5Depa men o Heal h Technology, Technical Uni e si y o Denma k, 2800 Lyngby, Denma k; [email p o ec ed]
6Depa men o Ma e ials, Chemical and Polyme Enginee ing, Buein Zah a Technical Uni e si y,
Buein Zah a 34518-66391, Qaz in, I an; [email p o ec ed]
*Co espondence: s-hesa aki@me c.ac.i ; Tel.: +98-263-6204-1314
Abs ac :
In he p esen s udy, a no el composi e bone cemen based on calcium sul a e hemihyd a e
(CSH) and Mg, S -con aining bioac i e glass (BG) as solid phase, and solu ion o chi osan as liquid
phase we e de eloped. The phase composi ion, mo phology, se ing ime, injec abili y, iscosi y,
and cellula esponses o he composi es wi h a ious con en s o BG (0, 10, 20, and 30 w .%) we e
in es iga ed. The pu e calcium sul a e cemen was se a app oxima ely 180 min, whe eas he se ing
ime was d as ically dec eased o 6 min by eplacing 30 w .% glass powde o CSH in he cemen
solid phase. BG changed he mic oscopic mo phology o he se cemen and dec eased he size and
compac ion o he p ecipi a ed gypsum phase. Replacing he CSH phase wi h BG inc eased injec ion
o ce o he p oduced cemen ; howe e , all he cemen s we e injec ed a a nea ly cons an o ce,
lowe han 20 N. The iscosi y measu emen s in oscilla o y mode de e mined he shea - hinning
beha io o he pas es. Al hough he iscosi y o he pas es inc eased wi h inc easing BG con en ,
i was in luenced by he equency ex en . Pu e calcium sul a e cemen exhibi ed some ansien
cy o oxici y on human-de i ed bone mesenchymal s em cells and i was compensa ed by in oducing
BG phase. Mo eo e , BG imp o ed he cell p oli e a ion and mine aliza ion o ex acellula ma ix as
shown by calcein measu emen s. The esul s indica e he injec able composi e cemen comp ising
70 w .% CSH and 30 w .% Mg, S -doped BG has be e se ing, mechanical and cellula beha io s and
hence, is a po en ial candida e o bone epai , howe e mo e animal and human clinical e alua ions
a e essen ial.
Keywo ds:
calcium sul a e; bioac i e glass; heology; bone cemen ; chi osan; S -doped bioma e ials
1. In oduc ion
I is he s anda d p ocedu e o su geons o use dono bone o syn he ic ma e ials o
ill up osseous de ec s le by p ocedu es like a h oplas y, bone umo emo al, in ec ion,
ac u e, o e en auma. Al e na i es o allog a s and au og a s ha e been a ailable
o many decades in he o m o syn he ic polyme s, ce amics, and polyme /ce amics
composi es. These bone subs i u es and ille s a e a ailable in di e en shapes, including
bulk pieces, injec able non-cemen pas es, and moldable cemen - ype o mula ions [1–6].
Bone subs i u es cemen s (BSCs) a e composed o powde and app op ia e solu ion
phases and by mixing, hey gi e a shapeable and sel -se ing pas e. The e o e, hese BSC
Biomedicines 2023,11, 2833. h ps://doi.o g/10.3390/biomedicines11102833 h ps://www.mdpi.com/jou nal/biomedicines
Biomedicines 2023,11, 2833 2 o 17
bioma e ials mus ha e good heological and injec able p ope ies p io o sel -se ing o
allow he su geon o deli e bone cemen s pas e h ough a needle o cannula in o bone
de ec s wi h i egula shapes and wi hou p ope access du ing he su gical p ocess.
Calcium sul a e (CS), i-calcium phospha e, and hyd oxyapa i e a e some ypical
o ms o bone ille s used he apeu ically [
7
,
8
]. Among bone cemen s, calcium sul a e
cemen is known as a well- ole a ed, as se ing, apidly and comple ely bio eso bable
ma e ial which no only is able o be used as bone ille , bu can also be used as local
deli e y media o he apeu ic agen s [
9
]. Despi e he a o able p ope ies, calcium sul a e
su e s om some weak poin s such as he apid eso p ion a e and disabili y in chemical
bonding wi h adjacen issues (bioaci i y) and weak mechanical s eng h [
10
,
11
]. All he
abo e-men ioned poin s ema kably limi he clinical applica ions o CS cemen s. To
o e come he abo e-men ioned challenges, esea che s ha e enginee ed calcium sul a e
composi e wi h mo e sui able p ope ies using di e en ypes o addi i es such as na u al
polyme s (e.g., sodium algina e, gela in, collagen) and bioac i e ino ganic ma e ials such
as hyd oxyapa i e and bioac i e glass (BG) [
12
–
16
]. I has been shown ha inco po a ion
o some polyme s in o CS cemen ma ix could enhance he injec abili y, cohesion, and
mechanical p ope ies [17].
Beside he polyme ic addi i es, he p esence o ino ganic nanopa icles such as hy-
d oxyapa i e and BGs can o e he physical and mechanical p ope ies and egula e he
deg ada ion a e and he bioac i i y o CS cemen s, owing o hei dissolu ion p oduc s,
which ac as igge ing os eop ogeni o cells a he gene ic le el. In one s udy [
18
], he
addi ion o i anium-doped nano-hyd oxyapa i e in CS-based composi es enhanced he
se ing ime, mechanical s eng h, and bioac i i y. Ano he s udy examined cemen pas es
composed o h ee biopolyme s o gela in/algina e/chond oi in sul a e and
α
-CSH and
calcium-de icien hyd oxyapa i e (CDHA) [
17
]. Thei esul s demons a ed he composi e
pas es exhibi ed op imal heological beha io s, good handling p ope ies as well as ap-
p op ia e an i-washou cha ac e is ics. BGs in combina ion wi h a ious polyme s ha e
also been in oduced in o he CS cemen ; he biological, physicochemical, and mechanical
ea u es o he acqui ed cemen pas es we e in es iga ed [
9
,
17
,
19
]. BG can o m a chemical
bond wi h li ing issues and p omo es bone o ma ion in a eas dis an om he implan
si e [20,21].
Chi osan is an app op ia e na u al polyme wi h dis inc i e ad an ages ha can be
inco po a ed wi h cemen s o p o ide adequa e physical and biological p ope ies. I
is hemos a ic, ungis a ic, and biodeg adable, and exhibi s an i umo , immunoadju an
cha ac e is ics. I can bind o human and mic obial cells, and accele a es he gene a ion
o os eoblas , which is esponsible o bone o ma ion [
22
]. In addi ion, chi osan shows
an imic obial p ope y agains G am-posi i e and nega i e bac e ia. The an ibac e ial
ac i i y exhibi s a conside able dependence on bo h he ca ionic cha ge and he molecula
weigh [
23
]. These di e se ea u es o chi osan make i easy o hem o be used in sca olds,
nanopa icles, beads, mic opa icles, nano ibe s, memb anes, and o he o ms [22].
One o he impo an cha ac e is ics o CS-based cemen s is injec abili y. Injec abili y
p o ides non-in asi e ans e o cemen i ious ma e ial in o he bone ca i ies h ough a
needle o cannula, ollowed by quick in si u se ing o he illing pas e [
24
]. Since low
beha io is a key ac o in injec able sys ems, he heological cha ac e is ics o he cemen
pas es should be ca e ully assessed. Zima e al. [
25
] conduc ed esea ch on he heology o
CS cemen s, emphasizing he in luence o he ion p esence on mul iple in lec ions in he
iscosi y- ime diag am.
In he p esen s udy, a no el bone cemen based on calcium sul a e, Mg, S -con aining
bioac i e glass, and chi osan was de eloped. A se ies o cemen s we e p epa ed by mixing
a ious amoun s o sol-gel de i ed Mg, S -doped BG pa icles, and
α
-CS as powde phase
and chi osan solu ion as liquid phase. The heological beha io , injec abili y, and he
co ela ion be ween injec ion o ce and iscosi y we e de e mined. The phase composi ion,
mo phology, se ing ime, and inally cellula cy ocompa ibili y we e also assessed.
Biomedicines 2023,11, 2833 3 o 17
2. Ma e ials and Me hods
2.1. Ini ial Powde s P epa a ion
Calcium sul a e hemihyd a e, CSH (CaSO
4·
0.5H
2
O, CAS-No. 10034-76-1, p oduc No.
12090) was pu chased om Me ck. Ci ic acid and medium molecula weigh chi osan
(CAS-No. 9012-76-4, p oduc No. 448877), C
12
H
24
N
2
O
9
, wi h M
W
: 200,000 g/mol, Deace y-
la ion
≥
75%, and pu i y > 98%, we e p epa ed om Sigma-Ald ich, Tau ki chen, Ge many.
Mg, S -doped bioac i e glass (BG) was also syn hesized acco ding o he p e iously de-
sc ibed me hod [
26
] in 64SiO
2
-30CaO-5P
2
O
5
sys em, in which he concen a ion o S and
Mg dopan s was 0.5 w .% o CaO componen .
2.2. Cemen Pas es P epa a ion
The CS–BG cemen s we e made by a combina ion o calcium sul a e hemihyd a e and
BG powde s in a ious mass a ios, and chi osan and ci ic acid solu ion wi h a powde
o liquid a io o 1.5. Table 1shows he composi ion o se e al CS–BG cemen s made a a
cons an P/L o 1.5 g/mL.
Table 1. Composi ion a ious CS–BG cemen s.
Code Powde Phase Liquid Phase
CSH
Solu ion o 19.2%
ci ic acid and 2%
chi osan (w/ )
CS100-BG0 100 0
CS90-BG10 90 10
CS80-BG20 80 20
CS70-BG30 70 30
2.3. Se ing Time, Po osi y and Comp essi e S eng h
The ini ial and inal se ing imes o he CS–BG pas es we e de e mined using a
s anda d es wi h Gillmo e needles, as desc ibed in ASTM C266–99 [27].
To de e mine he comp essi e s eng h, he cylind ical specimens we e ab ica ed
wi h a diame e o 6 mm and a heigh o 12 mm. A 24 h a e se ing, he specimens we e
ans e ed o a mechanical es ing de ice (SANTAM STM-20) and he comp ession es s
we e conduc ed wi h a a loading speed o 1 mm/min.
The o al po osi y (P) o he cemen s was calcula ed on he cylind ical specimens,
A chimedes me hod, whe e e hanol was used as medium [28].
2.4. Phase Composi ion
The phase composi ion o as-se cemen s was de e mined using X- ay di ac ome y,
XRD, (Philips PW 3710, Ams e dam, Ne he lands) wi h Cu-K
α
adia ion and a 1.54050 nm
X- ay wa eleng h, which was ope a ed a 40 kV and 40 mA wi h a s ep size o 0.02 and a
coun du a ion o 2 s/s ep.
2.5. SEM Obse a ion
The mo phological and chemical cha ac e is ics o CS–BG cemen s we e in es iga ed
using scanning elec on mic oscope (SEM) (TESCAN-XMU, VEGA II, B no–Kohou o ice,
Czech Republic) equipped wi h an ene gy dispe si e X- ay spec oscopy (EDS) analysis
uni . The map o S, Si, and N elemen s also de e mined he elemen al image analysis.
Mo eo e , he pa icle size analysis o he mic os uc u e was pe o med using Image J
so wa e e sion 1.0.8_112 and he g aphs we e d awn by ORIGIN PRO 2022.
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [
29
]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
Biomedicines 2023,11, 2833 4 o 17
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e
he mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine
(STM-20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion
cu e was plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien
(I) was calcula ed as
I = [(M0 −M)/M0] ×100 (1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e mea-
su ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac i e
glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e was
made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe pla e’s
cen e a p ede e mined in e als. The heological es began 2 min a e mixing he powde
and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing
o mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G0+ iG00 (2)
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
* =
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
0−i
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
00 (3)
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
0= G00/ω(4)
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
00 = G0/ω(5)
whe e
ω
is he equency in ads
−1
, G
0
is shea s o age modulus, G
00
is shea loss modulus,
G* is complex shea modulus,
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
00
is ou -o -phase iscosi y,
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
0
is dynamic (absolu e) iscosi y,
and
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y) (
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
0
)
is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [
30
,
31
]. Fi s ly, s ain
sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion (LVR).
Then, he dynamic equency sweep es was pe o med while main aining cons an s ain
(1%) and empe a u e (25 ◦C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo
in i o
cellula s udies, he samples we e imme sed in an an ibio ic solu ion o a
du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs)
was s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100
µ
L o he medium a
a densi y o 10
4
cells/well in 96-well cul u e pla e and we e incuba ed a 37
◦
C in a 95%
humidi ied a mosphe e o 95% ai and 5% CO
2
, o 24 h. Subsequen ly, sphe ical specimens
(wi h he same shape and su ace a ea) we e posi ioned in he cell cul u e medium and
incuba ed o 24, 48, and 72 h. The sample- ee cul u e pla e was used as con ol g oup.
A e incuba ion, he medium was e acua ed and 100
µ
L o se um ee MTT-con aining
cul u e medium (0.5 mg/mL) was added o each well, ollowed by a 4-h incuba ion a
37
◦
C. Finally, 100
µ
L o dime hyl sul oxide (Sigma, Ge many) was added o each well,
and cell iabili y was de e mined using a mic opla e eade (BioTek ELx800, Winooski,
Biomedicines 2023,11, 2833 5 o 17
VT, USA) a 570 nm. The pe cen age o iable cells (compa ed o he con ol g oup) we e
e alua ed and epo ed.
2.8.2. DAPI S aining
DAPI (4
0
,6-diamidino-2-phenylindole) is a luo escen s ain ha binds s ongly o
adenine– hymine ich egions in DNA and allows o obse ing he heal h o dea h o
he cell. Fo his pu pose, he hBMSCs was cul u ed on he cemen s, and on days o 1,
3, and 7, i was aken ou o he incuba o o s aining, he comple e cul u e medium
was emo ed and washed once, hen ixed wi h 4% (w/ ) pa a o maldehyde o 30 min.
The pa a o maldehyde was emo ed, he DAPI solu ion (D9542-1MG SIGMA) wi h a
concen a ion o 300 nM was pou ed on he cemen s and a e 5 min, i was obse ed unde
he luo escen mic oscope NIB-100F (NOVEL Co, Ningbo, China).
2.8.3. Calcein S aining
Cells we e cul u ed on he cemen acco ding o he s anda d p ocedu e. On he
desi ed day o s udy (days 14 and 21), cemen s con aining cells we e washed o he assay
wi h 1000
µ
L o a phospha e bu e o emo e any se um es e ase ac i i y ha may be
p esen in he g ow h media. Calcein powde (L468415 MERCK) was dissol ed in DMSO
and a wo king solu ion wi h a inal concen a ion o 2
µ
M was p epa ed. Nex , abou
200 mic oli e s was added o he samples, so i was co e ed and incuba ed a empe a u e
25
◦
C o 30 min. A e he passage o ime, i was obse ed wi h a luo escen mic oscope
NIB-100F (NOVEL Co. Ningbo, China Co.)
2.9. S a is ical Analysis
The da a we e collec ed om a leas h ee sepa a e expe imen s. The one-way ANOVA
was pe o med o he s a is ical analysis, and p alue < 0.05 was conside ed s a is ically
signi ican (* p< 0.05, ** p< 0.01, *** p< 0.001).
3. Resul s
3.1. Phase Composi ion
Figu e 1illus a es he XRD pa e ns o a ious CS–BG cemen s as well as s a ing
ma e ials (chi osan, CSH and BG) p esen ed o compa ison. The x- ay di ac ion pa e ns
o chi osan and bioac i e glass powde e eal cha ac e is ics o an amo phous phase. In he
pa e ns o se cemen CS100-BG0, he un eac ed CSH is he p edominan phase and some
mino gypsum p oduc is obse ed (PDF-Numbe 33-0311). The same phase composi ion
can also be seen in he di ac ion pa e ns o BG-con aining cemen s, excep an amo phous
backg ound seen due o he p esence o BG phase. The amo phous-like backg ound in he
XRD pa e ns o hese samples may also be ela ed o he o ma ion o amo phous chela e
(complex) p oduced om he chemical eac ion o Ca ions (deli e ed om BG and CSH)
and ci ic acid and/o chi osan molecules.
Biomedicines 2023,11, 2833 6 o 17
Biomedicines 2023, 11, 2833 6 o 17
Figu e 1. XRD pa e n o ini ial powde s and CS–BG cemen s.
3.2. Se ing Time
The ini ial and inal se ing imes, comp essi e s eng h and po osi y o he cemen s
a e gi en in Table 2. F om he e, we can see ha while pu e calcium sul a e hemihyd a e
combined wi h pu e dis illed wa e se s a abou 35 min, he chi osan-con aining calcium
sul a e cemen exhibi s a e y long se ing ime. The C100-BG0 is also weak in e ms o
comp essi e s eng h. A signi ican dec ease in se ing ime was obse ed when BG was
included in o he composi ion. He e, we obse ed a signi ican di e ence be ween he se -
ing imes o he a ious composi ions, especially o CS70-BG30. The comp essi e
s eng h is d as ically imp o ed in high con en s o BG. Also, he po osi y is in he ange
o 30–60% and excep o CS90-BG10, adding BG dec eases he o al po e con en .
Table 2. Ini ial and inal se ing imes, comp essi e s eng h, and o al po osi y o calcium sul a e
cemen s con aining di e en amoun s o BG.
Sample
Ini ial Se ing
Time (min)
Final Se ing
Time (min)
Comp essi e
S eng h (MPa)
Po osi y
(%)
Cemen made o pu e CS and dis-
illed wa e
25 ± 3
35 ± 3
-
-
Cemen made o pu e CS and ci ic
acid
180 ± 15
240 ± 20
-
-
CS100-BG0
>300
>600
0.29 ± 0.02
50 ± 2
CS90-BG10
60 ± 5
150 ± 20
0.20 ± 0.07
61 ± 3
CS80-BG20
32 ± 5
120 ± 10
1.25 ± 0.11
38 ± 1
CS70-BG30
6 ± 1
20 ± 4
4.25 ± 0.85
36 ± 2
3.3. SEM Obse a ion
Mic os uc u es o in es iga ed CS–BG cemen s a e shown in Figu es 2 and 3. Figu e
2 (A1–D3) depic s he SEM images and ela ed EDX analysis o CS–BG cemen s. The e-
sul s o pa icle size dis ibu ion pe o med by Image J so wa e a e shown in he inse o
each ela ed image. CS100-BG0 has po ous s uc u e mo phology consis ing o in e lock-
ing hexagonal lake-like c ys als. Ca, S, and O elemen s a e ound in he ela ed EDXA
image o CS100-BG0. A mono-modal pa icle size dis ibu ion wi h a e age size o 7.9 μm
is obse ed. By adding BG o calcium sul a e, he ollowing changes in he mic os uc u e
a e obse ed: (i) Fine glass pa icles embedded wi hin a monoli hic-shaped phase ha
co e ed he la ge block-like calcium sul a e c ys als a e seen; (ii) The mo phology and
shape o lakes changes om egula hexagonal o ela i ely shapeless blocks pa icles
Figu e 1. XRD pa e n o ini ial powde s and CS–BG cemen s.
3.2. Se ing Time
The ini ial and inal se ing imes, comp essi e s eng h and po osi y o he cemen s
a e gi en in Table 2. F om he e, we can see ha while pu e calcium sul a e hemihyd a e
combined wi h pu e dis illed wa e se s a abou 35 min, he chi osan-con aining calcium
sul a e cemen exhibi s a e y long se ing ime. The C100-BG0 is also weak in e ms o
comp essi e s eng h. A signi ican dec ease in se ing ime was obse ed when BG was
included in o he composi ion. He e, we obse ed a signi ican di e ence be ween he
se ing imes o he a ious composi ions, especially o CS70-BG30. The comp essi e
s eng h is d as ically imp o ed in high con en s o BG. Also, he po osi y is in he ange o
30–60% and excep o CS90-BG10, adding BG dec eases he o al po e con en .
Table 2.
Ini ial and inal se ing imes, comp essi e s eng h, and o al po osi y o calcium sul a e
cemen s con aining di e en amoun s o BG.
Sample Ini ial Se ing
Time (min)
Final Se ing
Time (min)
Comp essi e
S eng h (MPa)
Po osi y
(%)
Cemen made o
pu e CS and
dis illed wa e
25 ±3 35 ±3 - -
Cemen made o
pu e CS and
ci ic acid
180 ±15 240 ±20 - -
CS100-BG0 >300 >600 0.29 ±0.02 50 ±2
CS90-BG10 60 ±5 150 ±20 0.20 ±0.07 61 ±3
CS80-BG20 32 ±5 120 ±10 1.25 ±0.11 38 ±1
CS70-BG30 6 ±1 20 ±4 4.25 ±0.85 36 ±2
3.3. SEM Obse a ion
Mic os uc u es o in es iga ed CS–BG cemen s a e shown in Figu es 2and 3. Figu e 2
(A1–D3) depic s he SEM images and ela ed EDX analysis o CS–BG cemen s. The esul s
o pa icle size dis ibu ion pe o med by Image J so wa e a e shown in he inse o each
ela ed image. CS100-BG0 has po ous s uc u e mo phology consis ing o in e locking
hexagonal lake-like c ys als. Ca, S, and O elemen s a e ound in he ela ed EDXA image
o CS100-BG0. A mono-modal pa icle size dis ibu ion wi h a e age size o 7.9
µ
m is
obse ed. By adding BG o calcium sul a e, he ollowing changes in he mic os uc u e a e
obse ed: (i) Fine glass pa icles embedded wi hin a monoli hic-shaped phase ha co e ed
Biomedicines 2023,11, 2833 7 o 17
he la ge block-like calcium sul a e c ys als a e seen; (ii) The mo phology and shape o
lakes changes om egula hexagonal o ela i ely shapeless blocks pa icles (pa icula ly
o CS90-BG10 and CS80-BG20); (iii) In CS70-BG30, a lake-like mo phology wi h educed
hickness and pa icle size is obse ed. The pa icle size dis ibu ion de e mines ha when
BG is added, a bimodal size dis ibu ion is ound. In he au ho s’ opinion, he smalle
a e age size ela es o emaining BG ha did no eac wi h chi osan molecules, whe eas he
la ge a e age size is a ibu ed o calcium sul a e c ys als. By inc easing he BG con en , he
a e age size o calcium sul a e c ys als dec eases, so ha in CS70-BG30, he bimodal g aph
ends o a mono-modal. In he EDXA pa e ns o BG-added CS, in addi ion o Ca, S, and O,
he P and Si elemen s a e also ound. The peaks o S o e lap wi h P and Si, meanwhile he
de ec ion o Mg and S peaks is impossible because o he e y low concen a ion o hem
in he glass composi ion. I should be no ed ha he EDXA is no a quan i a i e analysis
and he con en o he elemen s should no be judged by he in ensi y o he peaks.
Biomedicines 2023, 11, 2833 7 o 17
(pa icula ly o CS90-BG10 and CS80-BG20); (iii) In CS70-BG30, a lake-like mo phology
wi h educed hickness and pa icle size is obse ed. The pa icle size dis ibu ion de e -
mines ha when BG is added, a bimodal size dis ibu ion is ound. In he au ho s’ opinion,
he smalle a e age size ela es o emaining BG ha did no eac wi h chi osan mole-
cules, whe eas he la ge a e age size is a ibu ed o calcium sul a e c ys als. By inc eas-
ing he BG con en , he a e age size o calcium sul a e c ys als dec eases, so ha in CS70-
BG30, he bimodal g aph ends o a mono-modal. In he EDXA pa e ns o BG-added CS,
in addi ion o Ca, S, and O, he P and Si elemen s a e also ound. The peaks o S o e lap
wi h P and Si, meanwhile he de ec ion o Mg and S peaks is impossible because o he
e y low concen a ion o hem in he glass composi ion. I should be no ed ha he EDXA
is no a quan i a i e analysis and he con en o he elemen s should no be judged by he
in ensi y o he peaks.
Figu e 2. SEM and he co esponding EDS images o CS100-BG0 (A1–A3), CS90-BG10 (B1–B3),
CS80-BG20 (C1–C3), and CS70-BG30 (D1–D3) cemen s.
Figu e 2.
SEM and he co esponding EDS images o CS100-BG0 (
A1
–
A3
), CS90-BG10 (
B1
–
B3
),
CS80-BG20 (C1–C3), and CS70-BG30 (D1–D3) cemen s.
Biomedicines 2023,11, 2833 8 o 17
Biomedicines 2023, 11, 2833 8 o 17
Figu e 3 depic s he SEM elemen al mapping o CS–BG samples o elemen al dis i-
bu ion. Blue spo s indica e he p esence o Si as he p incipal elemen in he BG s uc u e.
G een do s ep esen S (Sul u ) as ep esen a i e o CaSO4, whe eas yellow do s symbol-
ize N (ni ogen) as ing edien o chi osan. The SEM o he su aces o he samples (p o-
ided in he le side o he map images) is in ag eemen wi h hose shown in Figu e 2.
The addi ion o BG o calcium sul a e al e s he mic os uc u e, and when he pe cen age
o bioac i e glass eaches 30 pe cen , he mic os uc u e o calcium sul a e-bioac i e glass
changes gene ally. The su ace o CS–BGs shows a signi ican di e ence when he BG
con en inc eased. In CS90-BG10 and CS80-BG20, la ge da k a eas a e obse ed and he Si
dis ibu ion seems he e ogeneous. I can be ela ed o he inhomogenei y o he su ace
c ea ed by he la ge po es. In he case o CS70-BG30, wi h a highe pe cen o BG, he
mic os uc u e was changed and ine lakes along wi h he emaining CSH eac an s a e
obse ed. Also, he SEM image e eal ha he mic os uc u e o he CS70-BG30 con ains
a ine pa icle, mo e educed po osi y, dense and smoo he su aces. The e o e, he dis-
ibu ion o blue do s seems mo e homogenous.
Figu e 3. Su ace mo phology and elemen al dis ibu ion o S, Si, and N.
3.4. Injec abili y
Figu e 4A shows he injec ion cu es o cemen s wi h di e en con en s o BG. A he
beginning poin o es , a sudden inc ease in ex uding o ce is obse ed o all cemen s.
This was also obse ed in o he esea ch. In a pilo es , he o ce-displacemen diag am
o emp y sy inge was checked. In his si ua ion, no o e shoo was obse ed a he s a o
he es . I de e mines ha he sy inge wall-plunge ic ion is negligible and he beginning
Figu e 3. Su ace mo phology and elemen al dis ibu ion o S, Si, and N.
Figu e 3depic s he SEM elemen al mapping o CS–BG samples o elemen al dis i-
bu ion. Blue spo s indica e he p esence o Si as he p incipal elemen in he BG s uc u e.
G een do s ep esen S (Sul u ) as ep esen a i e o CaSO
4
, whe eas yellow do s symbolize
N (ni ogen) as ing edien o chi osan. The SEM o he su aces o he samples (p o ided in
he le side o he map images) is in ag eemen wi h hose shown in Figu e 2. The addi ion
o BG o calcium sul a e al e s he mic os uc u e, and when he pe cen age o bioac i e
glass eaches 30 pe cen , he mic os uc u e o calcium sul a e-bioac i e glass changes gen-
e ally. The su ace o CS–BGs shows a signi ican di e ence when he BG con en inc eased.
In CS90-BG10 and CS80-BG20, la ge da k a eas a e obse ed and he Si dis ibu ion seems
he e ogeneous. I can be ela ed o he inhomogenei y o he su ace c ea ed by he la ge
po es. In he case o CS70-BG30, wi h a highe pe cen o BG, he mic os uc u e was
changed and ine lakes along wi h he emaining CSH eac an s a e obse ed. Also, he
SEM image e eal ha he mic os uc u e o he CS70-BG30 con ains a ine pa icle, mo e
educed po osi y, dense and smoo he su aces. The e o e, he dis ibu ion o blue do s
seems mo e homogenous.
3.4. Injec abili y
Figu e 4A shows he injec ion cu es o cemen s wi h di e en con en s o BG. A he
beginning poin o es , a sudden inc ease in ex uding o ce is obse ed o all cemen s.
This was also obse ed in o he esea ch. In a pilo es , he o ce-displacemen diag am
o emp y sy inge was checked. In his si ua ion, no o e shoo was obse ed a he s a
o he es . I de e mines ha he sy inge wall-plunge ic ion is negligible and he
Biomedicines 2023,11, 2833 9 o 17
beginning o e shoo co esponds o he o ce equi ed o o e come he pas e-sy inge
wall ic ion caused by hyd aulic p essu e inside he sy inge [
29
,
32
]. A e ha , all he
cemen s a e injec ed wi h a nea ly cons an o ce, lowe han 20 N. O e all, i is ound ha
he composi es wi h highe BG con en exhibi highe injec ion o ce. No il e -p essing
phenomenon is obse ed du ing he injec ion. I seems ha he ex usion o ce dec eased
a e 5 mm o displacemen . I was ound ha he pas e was cohe en and slippe y due he
p esence o chi osan. The au ho s sugges , when a olume o he pas e is ex uded, lowe
o ce is equi ed o ex ude he emaining pas e. Thus, a dec eased p o ile is seen o he
ex usion by sy inge displacemen .
Biomedicines 2023, 11, 2833 9 o 17
o e shoo co esponds o he o ce equi ed o o e come he pas e-sy inge wall ic ion
caused by hyd aulic p essu e inside he sy inge [29,32]. A e ha , all he cemen s a e
injec ed wi h a nea ly cons an o ce, lowe han 20 N. O e all, i is ound ha he compo-
si es wi h highe BG con en exhibi highe injec ion o ce. No il e -p essing phenomenon
is obse ed du ing he injec ion. I seems ha he ex usion o ce dec eased a e 5 mm o
displacemen . I was ound ha he pas e was cohe en and slippe y due he p esence o
chi osan. The au ho s sugges , when a olume o he pas e is ex uded, lowe o ce is e-
qui ed o ex ude he emaining pas e. Thus, a dec eased p o ile is seen o he ex usion
by sy inge displacemen .
Since in he injec abili y es , he pas e is ex uded a a nea ly cons an a e, and he
o ce equi ed o he ex usion is e alua ed, i may be possible o c ea e an equi alence
be ween he eal pa o he iscosi y (ղ’) and he o ce equi ed o injec ion. I means ha
ղ’ can be conside ed a c i e ion o he o ce equi ed o injec ion.
Figu e 4B shows he eal pa o he complex iscosi y (ղ’) o a ious cemen s as a
unc ion o ω calcula ed om Equa ion (4). In o he wo ds, he e is a di ec co ela ion
be ween he injec ion o ce and (ղ’). I can be seen ha he s a ing poin s o ղ’ inc ease
wi h in oducing conside able amoun o BG, which is consis en wi h he injec abili y
cu e o cemen s. I means ha a highe injec ion o ce is equi ed. Mo eo e , a low e-
quencies ange (ω = 1 − 8 s−1), wi h inc easing ω, a sha p dec ease in he eal pa o is-
cosi y is obse ed (especially CS70-BG30) and he o ce equi ed he injec ion o be con-
inued dec eases.
Figu e 4. (A) The injec ion cu es o di e en cemen s shown as applied o ce s. plunge displace-
men . (B) The eal pa o he complex iscosi y (ղ’) o a ious cemen s as a unc ion o ω.
3.5. Viscosi y
Figu e 5 shows he complex iscosi y (ղ*) o he pas e as a unc ion o ω. The complex
iscosi y o cemen s wi h highe pe cen o bioac i e glass is mo e sensi i e o angula
equency.
The angula equency alue o 10 s−1 is he in e sec ion poin o ղ*-ω cu es. In o he
wo ds, a ω < 10 s−1, cemen s wi h highe BG con en ha e a highe ղ* alue and he cemen
CS70-BG30 exhibi s he highes complex iscosi y. A equencies highe han 10 s−1, he
iscosi y beha es comple ely di e en and samples wi h mo e bioac i e glass con en
ha e a lowe iscosi y. In his case, he highes iscosi y ela es o he glass- ee cemen .
In ac , mo e in e nal s uc u es can be o med in pas es con aining bioac i e glass, and
when he equency exceeds 10 s−1, hese s uc u es a e b oken leading o a sudden de-
c ease o iscosi y. In he case o hese cemen s, wi h an inc ease in equency (mo e han
100 s−1), he iscosi y inc eases again and shea hickening beha io is obse ed. I means
ha he des oyed in e nal s uc u es (links) a e e o med due o he in ensi e s esses.
Figu e 4.
(
A
) The injec ion cu es o di e en cemen s shown as applied o ce s. plunge displace-
men . (B) The eal pa o he complex iscosi y (
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
’) o a ious cemen s as a unc ion o ω.
Since in he injec abili y es , he pas e is ex uded a a nea ly cons an a e, and he
o ce equi ed o he ex usion is e alua ed, i may be possible o c ea e an equi alence
be ween he eal pa o he iscosi y (
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
’) and he o ce equi ed o injec ion. I means ha
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
’ can be conside ed a c i e ion o he o ce equi ed o injec ion.
Figu e 4B shows he eal pa o he complex iscosi y (
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
’) o a ious cemen s as a
unc ion o
ω
calcula ed om Equa ion (4). In o he wo ds, he e is a di ec co ela ion
be ween he injec ion o ce and (
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
’). I can be seen ha he s a ing poin s o
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
’ inc ease wi h
in oducing conside able amoun o BG, which is consis en wi h he injec abili y cu e o
cemen s. I means ha a highe injec ion o ce is equi ed. Mo eo e , a low equencies
ange (
ω
= 1
−
8 s
−1
), wi h inc easing
ω
, a sha p dec ease in he eal pa o iscosi y
is obse ed (especially CS70-BG30) and he o ce equi ed he injec ion o be con inued
dec eases.
3.5. Viscosi y
Figu e 5shows he complex iscosi y (
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
*) o he pas e as a unc ion o
ω
. The complex
iscosi y o cemen s wi h highe pe cen o bioac i e glass is mo e sensi i e o angula
equency.
The angula equency alue o 10 s
−1
is he in e sec ion poin o
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
*-
ω
cu es. In
o he wo ds, a
ω
< 10 s
−1
, cemen s wi h highe BG con en ha e a highe
Biomedicines 2023, 11, 2833 4 o 17
2.6. Injec abili y
An ex usion es was used o assess he pas e injec abili y [29]. A e 1-min mixing
p ocedu e, he ob ained pas e was placed in o a comme cial 3 mL sy inge (A aPezeshk
Co., Teh an, I an) wi h cannula leng h and ip diame e o 10 mm and 2 mm, espec i ely.
The pas e was exe ed in sy inge ube ( he leng h o illed pas e in he ube was 30 mm)
and ex uded om he sy inge (held igidly in a ix u e) a p ede e mined imes a e he
mixing p ocess was comple ed by applying o ce using a uni e sal es ing machine (STM-
20, San am Co., Teh an, I an) a a c osshead speed o 30 mm/min. The injec ion cu e was
plo ed as injec ion o ce s. plunge displacemen . The injec abili y coe icien (I) was cal-
cula ed as
I = [(M0 − M)/M0] × 100
(1)
whe e M0 is he ini ial mass o he composi e in he sy inge and M is he mass le inside
he sy inge a e ex usion.
2.7. The Viscosi y Measu emen s
The Rheome e (An on Paa , Physica MC-R301, G az, Aus ia) wi h pla e–pla e meas-
u ing geome y was used o examine he heological p ope ies o calcium sul a e-bioac-
i e glass pas es (pla e diame e o 25 mm). Fo his measu emen , a homogeneous pas e
was made du ing 1 min and he esul an pas e was subsequen ly applied o he lowe
pla e’s cen e a p ede e mined in e als. The heological es began 2 min a e mixing
he powde and liquid phases a bo h oscilla o y and o a ion modes.
A modes (low) ampli ude sinusoidal oscilla ion s ain was i s ly applied o he pas e
in a dynamic oscilla ion es . The complex iscosi y can be s a ed using he ollowing o -
mula due o he phase di e ence be ween wo sinusoidal wa es.
G* = G′ + iG″
(2)
ղ* = ղ′ − iղ″
(3)
ղ’ = G″/ω
(4)
ղ” = G′/ω
(5)
whe e ω is he equency in ads−1, G′ is shea s o age modulus, G″ is shea loss modulus,
G* is complex shea modulus, ղ″ is ou -o -phase iscosi y, ղ′ is dynamic (absolu e) iscos-
i y, and ղ* is complex iscosi y. The eal pa o he complex iscosi y (dynamic iscosi y)
(ղ′) is a c i e ion o o ce equi ed o make a luid low a a ce ain speed [30,31]. Fi s ly,
s ain sweep mode measu emen was pe o med o iden i y he linea iscoelas ic egion
(LVR). Then, he dynamic equency sweep es was pe o med while main aining con-
s an s ain (1%) and empe a u e (25 °C).
In o a o y mode, he iscosi y– ime cu e was also d awn a a cons an shea a e o
1 s−1 o iden i y he a ia ion o he pas e iscosi y as a unc ion o ime.
2.8. The Cell S udies
2.8.1. Cell Su i al, Viabili y, and G ow h
Fo in i o cellula s udies, he samples we e imme sed in an an ibio ic solu ion o
a du a ion o 30 min; a e wa ds, hey we e insed in phospha e-bu e ed saline (PBS), and
subjec ed o ul a iole i adia ion (UV) o a pe iod o 15 min.
The cy o oxic e ec o cemen s on human bone mesenchymal s em cells (hMSCs) was
s udied by 3-(4,5-dime hyl hiazol-2)-2,5-diphenyl e azolium b omide assay (MTT;
Sigma-Ald ich, Tau ki chen, Ge many). The cells we e seeded in 100 μL o he medium a
a densi y o 104 cells/well in 96-well cul u e pla e and we e incuba ed a 37 °C in a 95%
* alue and
he cemen CS70-BG30 exhibi s he highes complex iscosi y. A equencies highe han
10 s
−1
, he iscosi y beha es comple ely di e en and samples wi h mo e bioac i e glass
con en ha e a lowe iscosi y. In his case, he highes iscosi y ela es o he glass- ee
cemen . In ac , mo e in e nal s uc u es can be o med in pas es con aining bioac i e glass,
and when he equency exceeds 10 s
−1
, hese s uc u es a e b oken leading o a sudden
dec ease o iscosi y. In he case o hese cemen s, wi h an inc ease in equency (mo e han
100 s
−1
), he iscosi y inc eases again and shea hickening beha io is obse ed. I means
ha he des oyed in e nal s uc u es (links) a e e o med due o he in ensi e s esses.
Biomedicines 2023,11, 2833 16 o 17
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Disclaime /Publishe ’s No e:
The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
