den al ma e ials 3 2 ( 2 0 1 6 ) 264–277
A ailable online a www.sciencedi ec .com
ScienceDi ec
jou nal homepage: www.in l.else ie heal h.com/jou nals/dema
Poly(p opylene glycol) and u e hane
dime hac yla es imp o e con e sion o den al
composi es and e eal complexi y o
cy ocompa ibili y es ing
Nick J. Wal e sa,1, Wendy Xiaa, Vehid Saliha,b, Paul F. Ashleyc,
Anne M. Younga,∗
aDi ision o Bioma e ials & Tissue Enginee ing, UCL Eas man Den al Ins i u e, 256 G ay’s Inn Road, London WC1X
8LD, UK
bPlymou h Uni e si y Peninsula Schools o Medicine and Den is y, Po land Squa e, D ake Ci cus, Plymou h, De on
PL4 8AA, UK
cPaedia ic Den is y, UCL Eas man Den al Ins i u e, 256 G ay’s Inn Road, London WC1X 8LD, UK
a i c l e i n o
A icle his o y:
Recei ed 7 Janua y 2015
Recei ed in e ised o m
20 May 2015
Accep ed 30 No embe 2015
Keywo ds:
Den al composi e
Den al ma e ial
Monome
Cy ocompa ibili y
Deg ee o con e sion
Polyme iza ion sh inkage
PPGDMA
TEGDMA
UDMA
Bis-GMA
a b s a c
Objec i es. To de e mine he e ec s o a ious monome s on con e sion and cy ocompa i-
bili y o den al composi es and o imp o e hese p ope ies wi hou de imen ally a ec ing
mechanical p ope ies, dep h o cu e and sh inkage.
Me hods.Composi es con aining u e hane dime hac yla e (UDMA) o bisphenol A glycidyl
me hac yla e (Bis-GMA) wi h poly(p opylene glycol) dime hac yla e (PPGDMA) o ie hylene
glycol dime hac yla e (TEGDMA) we e cha ac e ized using he ollowing echniques: con e -
sion (FTIR a 1 and 4 mm dep hs), dep h o cu e (BS EN ISO 4049:2009 and FTIR), sh inkage
(BS EN ISO 17304:2013 and FTIR), s eng h and modulus (biaxial flexu al es ) and wa e
so p ion. Cy ocompa ibili y o composi es and hei liquid phase componen s was assessed
using h ee assays ( esazu in, WST-8 and MTS).
Resul s.UDMA significan ly imp o ed con e sion, BFS and dep h o cu e compa ed o Bis-
GMA, wi hou inc easing sh inkage. UDMA was cy o oxic a lowe concen a ions han
Bis-GMA, bu ex ac s o Bis-GMA-con aining composi es we e less cy ocompa ible han
o hose con aining UDMA. PPGDMA imp o ed con e sion and dep h o cu e compa ed o
TEGDMA, wi hou de imen ally a ec ing sh inkage. TEGDMA was shown by all assays o be
highly oxic. Resazu in, bu no WST-8 and MTS, sugges ed ha PPGDMA exhibi ed imp o ed
cy ocompa ibili y compa ed o TEGDMA.
∗Co esponding au ho . Tel.: +44 20 3456 2353.
E-mail add esses: nick.wal e s@u a.fi (N.J. Wal e s), anne.y[email p o ec ed] (A.M. Young).
1Cu en add ess: Adul S em Cell G oup, BioMediTech (Ins i u e o Biosciences & Medical Technology), FinnMedi 5, 33014 Uni e si y
o Tampe e, Finland.
h p://dx.doi.o g/10.1016/j.den al.2015.11.017
0109-5641/© 2015 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
265
Significance.
The
use
o
UDMA
and
PPGDMA
esul s
in
composi es
wi h
excellen
con e sion,
dep h
o
cu e
and
mechanical
p ope ies,
wi hou
inc easing
sh inkage.
Composi es
con-
aining
UDMA
appea
o
be
sligh ly
mo e
cy ocompa ible
han
hose
con aining
Bis-GMA.
These
monome s
may
he e o e
imp o e
he
ma e ial
p ope ies
o
den al
es o a ions,
pa -
icula ly
bulk
fill
ma e ials.
The
e ec
o
diluen
monome
on
cy ocompa ibili y
equi es
u he
in es iga ion.
©
2015
The
Au ho s.
Published
by
Else ie
L d.
This
is
an
open
access
a icle
unde
he
CC
BY
license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
1.
In oduc ion
Den al
composi es
a e
widely
used
as
den al
es o a i e
ma e-
ials
o
hei
high
s eng h
and
excellen
aes he ics.
These
consis
p ima ily
o :
a
liquid
phase
con aining
monome s
and
an
ini ia o
sys em
( ypically
pho o-ac i a ed);
a
fille
phase
( ypically
bo o-aluminosilica e
glass
pa icles),
which
p o-
ides
op imal
mechanical
and
aes he ic
p ope ies;
and
silane,
a
coupling
agen
which
enables
bonding
o
he
polyme
o
he
fille .
Bisphenol
A
glycidyl
me hac yla e
(Bis-GMA),
u e hane
dime hac yla e
(UDMA)
and
ie hylene
glycol
dime hac yla e
(TEGDMA)
a e
some
o
he
mos
commonly
used
monome s
in
den al
composi es.
Upon
pho oini ia ion,
hey
o m
a
c oss-
linked
polyme
ne wo k
which
ha dens
and
en aps
he
fille s
[1].
Composi es
a e
ypically
laye ed
in
inc emen s
and
his
is
ime-consuming
o
clinicians,
since
i
equi es
cu ing
each
inc emen
be o e
p oceeding
wi h
he
nex .
This
is
pa ic-
ula ly
an
issue
in
deepe
pos e io
ca i ies,
in
which
many
inc emen s
may
be
equi ed.
As
a
esul ,
bulk
fill
ma e ials
aim
o
o e come
his
issue
by
u ilizing
pho oini ia o s
which
a e
e ec i e
a
dep hs
o
4–5
mm,
as
well
as
monome s
wi h
low
double
bond
concen a ion
and,
in
some
cases,
monome s
which
a e
clea ed
du ing
polyme iza ion.
One
o
he
majo
limi ing
ac o s
o
composi es
is
he
close
in e play
be ween
deg ee
o
con e sion
and
o he
cha -
ac e is ics,
including
mechanical
p ope ies,
polyme iza ion
sh inkage,
wa e
so p ion
and
elu ion
o
oxic
componen s.
Since
he
le el
o
esidual
monome
in
a
composi e
a ec s
i s
biocompa ibili y,
mechanical
p ope ies
and
aes he ics
[2],
high
con e sion
is
ideal
o
op imiza ion
o
hese
p ope ies.
Con e sely,
howe e ,
high
con e sion
is
ypically
associa ed
wi h
high
olume ic
sh inkage.
In
he
pa ien ,
his
can
esul
in
mic obial
mic oleakage
(pene a ion
o
pa hogens
be ween
he
composi e
and
oo h),
ecu en
ca ies
and,
ul ima ely,
ailu e
o
he
es o a ion.
Monome s
wi h
low
double
bond
concen a ion
and
subsequen
low
sh inkage
a e
he e o e
op imal,
pa icula ly
in
he
case
o
bulk
fill
ma e ials,
due
o
he
la ge
olume
o
each
inc emen .
The
aim
o
he
p esen
esea ch
was
o
imp o e
he
con-
e sion,
s eng h
and
cy ocompa ibili y
o
den al
composi es
wi hou
de imen ally
a ec ing
polyme iza ion
sh inkage
o
dep h
o
cu e
by
ully
eplacing
Bis-GMA
wi h
UDMA
and
TEGDMA
wi h
PPGDMA.
This
is
due
o
he
g ea e
flexibili y
and
c oss-linking
densi y
o
UDMA
han
Bis-GMA
[3,4]
and
he
g ea e
flexibili y
and
significan ly
lowe
double
bond
concen-
a ion
o
PPGDMA
han
TEGDMA.
In
o de
o
in es iga e
he
e ec
o
each
monome
on
cy ocompa ibili y,
human
gingi al
fib oblas s
(HGF)
we e
cul u ed
in
solu ions
o
each
indi idual
liquid
phase
componen
a
a ying
concen a ions,
as
well
as
in
ex ac s
o
each
composi e
o mula ion.
Due
o
he
a iabil-
i y
be ween
di e en
cell
iabili y
assays,
which
a ises
om
he
a ge ing
o
di e en
enzymes
wi hin
he
cell,
and
he
ambigui y
o
he
widely
used
e m
‘biocompa ibili y’
[5],
h ee
assays
we e
compa ed.
The
null
hypo hesis
was
ha
eplace-
men
o
Bis-GMA
wi h
UDMA
and
TEGDMA
wi h
PPGDMA
would
ha e
no
e ec
on
hese
p ope ies.
2.
Ma e ials
and
me hods
2.1.
Ma e ials
2.1.1.
Composi e
pas e
p epa a ion
Mic ohyb id
den al
composi es
[1]
we e
p epa ed
using
10
w %
40
nm
umed
silica
(Ae osil
OX-50,
E onik
Indus-
ies
AG,
Essen,
Ge many)
and
90
w %
silane- ea ed
ba -
ium
bo o-aluminosilica e
glass
pa icles
o
a ious
sizes
(DMG
Chemisch-Pha mazeu ische
Fab ik
GmbH,
Hambu g,
Ge many).
These
we e
combined
wi h
ou
dime hac yla e-
based
liquid
phases.
The
liquid
phases
consis ed
o
bulk
monome
UDMA
(DMG)
o
Bis-GMA
(Polysciences
Inc.,
Eppel-
heim,
Ge many)
combined
wi h
diluen
monome
PPGDMA
(Polysciences)
o
TEGDMA
(DMG).
The
bulk
o
diluen
mola
a io
was
3.5:1.
The
liquid
phases
also
con ained
40
mM
(0.58–0.61
w %)
pho oini ia o
campho quinone
(CQ,
DMG),
60
mM
(0.82–0.86
w %)
co-ini ia o
N,N-dime hylaminoe hyl
me hac yla e
(DMAEMA,
Sigma–Ald ich,
Gillingham,
UK)
and
100
ppm
inhibi o
bu yla ed
hyd oxy oluene
(Sigma–Ald ich).
The
wo
phases
we e
combined
o
o m
composi e
pas es
using
a
cen i ugal
plane a y
mixe
(SpeedMixe ,
Hauschild
Enginee ing,
Hamm,
Ge many),
in
o de
o
minimize
ai
inco -
po a ion
and
ensu e
comple e
we ing
o
fille
pa icles.
The
powde
o
liquid
a io
(PLR)
was
kep
cons an
a
40
ol%
liq-
uid
(19.3–20.3
w %,
depending
on
liquid
phase
densi y).
Den al
composi es
we e
designa ed
abb e ia ions
based
on
hei
bulk
and
diluen
monome
con en :
UP,
UT,
BP
and
BT,
whe e
U,
B,
P
and
T
ep esen
UDMA,
Bis-GMA,
PPGDMA
and
TEGDMA,
espec i ely.
Comme cial
composi e
Fil ek
Z250
(3M
ESPE,
S .
Paul,
MN,
USA)
was
used
o
compa ison.
2.1.2.
Disc
specimen
p oduc ion
Excep
whe e
o he wise
s a ed,
disc-shaped
specimens
we e
moulded
by
applying
composi e
pas es
o
me al
ci clips
(in e nal
diame e
10.2
mm,
hickness
1
mm)
and
p essing
hem
be ween
wo
shee s
o
ace a e.
This
p e en s
oxygen
inhibi ion
du ing
polyme iza ion
and
expels
excess
pas e,
266
d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
ensu ing
simila
specimen
hickness.
Specimens
we e
pho o-
polyme ized
using
a
blue
ligh
emi ing
diode
cu ing
uni
wi h
a
wa eleng h
o
450–470
nm
and
powe
ou pu
wi h
pe iodic
le el
shi ing
o
1100–1330
mW/cm2(Demi
Plus,
Ke
Den al,
O ange,
CA,
USA),
in
di ec
con ac
wi h
he
ace a e.
The
cu -
ing
du a ion
o
each
es ing
me hod
a ied
and
is
de ailed
in
each
co esponding
sec ion.
2.2.
Me hods
2.2.1.
Handling
p ope ies
and
we -poin
de e mina ion
The
we -poin
o
each
liquid
was
de e mined
by
g adually
adding
a
small
quan i y
o
liquid
phase
o
a
known
mass
o
fille
phase
and
mixing,
un il
he
fille
was
su ficien ly
we ed
and
a
cohesi e
pas e
had
been
o med.
The
quan i y
o
liquid
phase
added
was
eco ded
and
no es
we e
made
ega ding
he
handling
p ope ies
o
each
o mula ion.
The
we -poin
( ol%)
was
hen
calcula ed
om
he
o al
mass
o
liquid
and
he
densi y
o
each
componen .
2.2.2.
Polyme iza ion
p ope ies
2.2.2.1.
Deg ee
o
con e sion.
The
con e sion
o
each
com-
posi e
was
de e mined
using
Fou ie
ans o m
in a ed
spec oscopy
(FTIR,
Sys em
2000,
Pe kinElme ,
See
G een,
UK).
Composi e
pas e
was
applied
o
ei he
a
single
o
ou
s acked
ci clips
o
he
same
dimensions.
These
we e
placed
on
he
diamond
o
an
a enua ed
o al
eflec ance
acces-
so y
(Golden
Ga e
ATR,
Specac
L d.,
O ping on,
UK)
and
co e ed
wi h
a
shee
o
ace a e.
A e
an
ini ial
spec um
o
he
uncu ed
composi e
had
been
ob ained,
spec a
we e
eco ded
con inuously
o
1000
s
(n
=
3).
The
specimens
we e
pho o-polyme ized
om
he
op
o
he
fi s
20
s.
Spec a
we e
eco ded
a
a
wa eleng h
ange
o
800–1800
cm−1and
esolu ion
o
8
cm−1.
Abso bance
p ofiles
we e
ob ained
a
1319
±
1
cm−1(C–O
s e ch
bond)
and
1334
±
2
cm−1(baseline)
and
used
o
calcula e
con e sion
a
1
mm
and
4
mm
dep hs
using
Eq.
(1):
C
=1
−A
Ao×
100
(1)
whe e
C
is
con e sion.
A and
A0a e
final
and
ini ial
abso bance
abo e
baseline,
espec i ely
[6].
2.2.2.2.
Polyme iza ion
sh inkage.
2.2.2.2.1.
Sh inkage
based
on
con e sion.
The
olume ic
sh inkage
o
composi es
was
calcula ed
om
he
con e sion
da a
using
Eq.
(2)
[7],
which
is
based
on
he
finding
ha
me hac yla e
es e s
ypically
unde go
olume ic
sh inkage
o
s
=
22.5
cm3/mol
upon
polyme iza ion
[8,9]:
S
=
100
×smCinixi
Wi (2)
whe e
S,
m,
C,
,
˙,
i,
n,
x
and
W
a e
sh inkage
( ol%),
monome
mass
ac ion
wi hin
composi e,
con e sion,
composi e
den-
si y,
sum
o
all
monome s
in
liquid
phase,
each
monome
in
liquid
phase,
numbe
o
C
C
bonds
pe
molecule,
mass
ac ion
o
monome
in
liquid
phase
and
molecula
mass,
espec i ely.
2.2.2.2.2.
Sh inkage
based
on
olume
change.
The
sh ink-
age
o
composi es
was
also
ob ained
by
measu ing
he
densi y
o
polyme ized
and
unpolyme ized
specimens
acco ding
o
BS
EN
ISO
17304:2013
[10].
This
echnique
uses
an
analy ical
bal-
ance
equipped
wi h
a
densi y
de e mina ion
appa a us
(AG
204
&
MS-DNY-43,
Me le
Toledo,
Beaumon
Leys,
UK)
and
is
based
on
A chimedes’
p inciple.
Disc
specimens
we e
cu ed
o
40
s
om
each
side
o
ensu e
comple e
con e sion.
Speci-
men
edges
we e
polished
o
emo e
loose
chips.
The
mass
o
h ee
cu ed
and
h ee
uncu ed
specimens
o
each
o mula ion
we e
measu ed
in
ai
and
unde
deionized
wa e .
Each
alue
o
mass
unde
wa e
was
a e aged
om
10
eadings.
The
sh inkage
o
composi es
and
hei
SD
we e
calcula ed
using
he
equa ions
p o ided
in
he
s anda d.
2.2.2.3.
Dep h
o
cu e.
The
dep h
o
cu e
o
composi es
(n
=
3)
was
measu ed
acco ding
o
BS
EN
ISO
4049:2009
[11].
B iefly,
composi e
pas e
was
applied
o
a
b ass
spli -mould
(in e nal
diame e
4
mm,
heigh
6
mm)
and
pho o-polyme ized
o
20
s
om
he
op.
The
specimen
was
emo ed
om
he
mould
and
a
plas ic
spa ula
was
used
o
emo e
any
uncu ed
ma e ial
om
he
bo om.
The
dep h
o
cu ed
ma e ial
was
measu ed
using
digi al
callipe s
o
an
accu acy
o
±0.01
mm
and
he
eading
was
hal ed,
as
equi ed
by
he
s anda d,
in
o de
o
gi e
a
alue
o
dep h
o
cu e.
2.2.2.4.
Wa e
so p ion.
In
o de
o
de e mine
wa e
so p ion,
disc
specimens
we e
cu ed
o
40
s
om
each
side
o
ensu e
maximum
con e sion.
The
mass
o
composi es
be o e
and
a e
imme sion
in
10
mL
deionized
wa e
o
one
week
was
hen
de e mined
using
an
analy ical
balance.
2.2.3.
Mechanical
p ope ies
Composi e
disc
specimens
(n
=
10)
we e
cu ed
o
40
s
om
each
side.
Specimens
we e
s o ed
d y
o
24
h,
be o e
place-
men
in
10
mL
deionized
wa e
a
37 ◦C
o
one
week.
They
we e
hen
es ed
using
a
biaxial
flexu al
es
(Au og aph
AGS-
X,
Shimadzu,
Mil on
Keynes,
UK),
wi h
a
2
kN
load
cell
and
ball-on- ing
jig
a
a
c oss-head
speed
o
1
mm/min,
un il
spec-
imen
ailu e.
Biaxial
flexu al
s eng h
(BFS,
MPa)
and
modulus
o
ensile
elas ici y
(E,
GPa)
we e
calcula ed
using
Eqs.
(3)
and
(4),
espec i ely,
BFS
=P
2(1
+
)0.485
ln a
+
0.52+
0.48(3)
E
=P
wc×ˇca2
3(4)
whe e
BFS,
P,
,

and
a
a e
biaxial
flexu al
s eng h,
ailu e
load
(N),
specimen
hickness
(mm),
Poisson’s
a io
(0.3)
and
jig
suppo
adius
(4
mm),
espec i ely,
and
E,
(P/Wc)
and
ˇc
a e
modulus,
g adien
o
elas ic
egion
and
cen e
deflec ion
unc ion
(0.5024)
[12],
espec i ely.
2.2.4.
Cy ocompa ibili y
2.2.4.1.
Cell
cul u e.
P ima y
HGF
we e
ob ained
om
a
com-
me cial
sou ce
(ScienCell
Resea ch
Labo a o ies,
Ca lsbad,
CA,
USA).
HGF
we e
cul u ed
unde
s anda d
condi ions
(37 ◦C,
95%
ai ,
5%
CO2,
95%
ela i e
humidi y)
in
Dulbecco’s
d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
267
modified
Eagle
medium
(DMEM,
Gibco,
Li e
Technologies,
Pais-
ley,
UK)
supplemen ed
wi h
10%
oe al
bo ine
se um
(Gibco)
and
1%
penicillin/s ep omycin
(PAA
Labo a o ies,
GE
Heal h-
ca e,
Chal on
S .
Giles,
UK).
Passage
numbe s
4–8
we e
used
o
cy ocompa ibili y
s udies.
2.2.4.2.
P epa a ion
o
es
solu ions.
Tes
solu ions
we e
p e-
pa ed
in
se um- ee
DMEM.
As
is
ypical
in
bioma e ials
es ing,
se um- ee
medium
was
used
in
o de
o
p e en
adso p ion
o
se um
p o eins
o
ma e ial
componen s.
Con-
ols
consis ed
o
se um- ee
DMEM.
2.2.4.2.1.
Liquid
phase
componen s.
Fi e
en- old
se ial
dilu ions
o
each
componen
we e
p epa ed
in
se um- ee
DMEM,
anging
om
0.01
o
100
mM
o
DMAEMA,
UDMA,
PPGDMA
and
TEGDMA,
and
0.001
o
10
mM
o
CQ
and
Bis-
GMA,
due
o
hei
lowe
solubili y.
The
solu ions
we e
s o ed
o
∼30
min
a
60 ◦C
and
hen
s i ed
using
a
s e ile
spa ula,
in
o de
o
aid
dissolu ion
o
componen s
wi h
low
solubili y,
pa icula ly
he
bulk
monome s.
2.2.4.2.2.
Composi e
ex ac s.
In
o de
o
p epa e
speci-
mens
o
ex ac
es ing,
a
1
mm
hick
ci clip
a op
a
shee
o
ace a e
was
filled
wi h
composi e
and
co e ed
wi h
ace a e.
A
u he
h ee
ci clips
we e
s acked
on
op
and
filled
wi h
composi e
and
co e ed
wi h
ace a e.
The
esul an
4
mm
deep
s ack
was
hen
pho o-polyme ized
o
20
s
om
he
op.
The
bo om
1
mm
hick
sec ion
was
emo ed
om
he
mould
and
incuba ed
in
650
␮L
se um- ee
DMEM
a
37 ◦C.
This
p o ided
an
ex ac ion
a io
o
1
mL/3
cm2su ace
a ea,
as
equi ed
by
ISO
10993-12:2009
[13].
Specimens
(n
=
3)
we e
agi a ed
a
100
pm
du ing
ex ac ion
(o bi al
shake ,
S ua
Scien ific,
S one,
UK).
A e
24
h,
specimens
we e
ans e ed
o
esh
medium
and
incuba ed
o
a
u he
6
days,
yielding
ex ac s
om
1
and
7-day
ime-poin s.
2.2.4.3.
Cy ocompa ibili y
assays.
HGF
we e
seeded
a
a
den-
si y
o
30,000
cells/cm2in
96
well
pla es
(n
=
3
pe
specimen,
ime-poin
and
assay).
A e
an
ini ial
24
h
seeding
pe iod,
cell
cul u e
medium
was
eplaced
wi h
100
␮L
o
he
specimen
(componen
solu ion,
composi e
ex ac
o
con ol).
The
solu-
ions
we e
ho oughly
mixed
using
a
o ex
mixe
(S ua
SA8,
Bibby
Scien ific,
S one,
UK)
in
o de
o
ensu e
e en
dispe sion
o
dissol ed
componen s.
Cy ocompa ibili y
was
assessed
a e
a
u he
24
h
o
cul u e
(48
h
ime-poin ),
in
acco -
dance
wi h
ISO
10993-5:2012
[14].
The
specimen
was
hen
eplaced
wi h
se um- ee
DMEM
and
cul u ed
o
a
u he
24
h
eco e y
pe iod
(72
h
ime-poin ).
Th ee
wa e -soluble
cell
me abolic
ac i i y
assays
we e
used
o
assess
cy ocom-
pa ibili y:
esazu in
(alama Blue
Cell
P oli e a ion
Assay,
AdB
Se o ech,
Bio-Rad
Labo a o ies
Inc.,
Hemel
Hemps ead,
UK);
wa e -soluble
e azolium
sal -8
(WST-8,
Cell
Coun ing
Ki -8,
Sigma–Ald ich);
and
3-(4,5-dime hyl hiazol-2-yl)-5-(3-
ca boxyme hoxyphenyl)-2-(4-sul ophenyl)-2H- e azolium
sal
(MTS,
CellTi e
96
AQueous One
Solu ion
Cell
P oli e a ion
Assay,
P omega,
Sou hamp on,
UK).
Medium
was
aspi a ed
om
he
cells
and
eplaced
wi h
100
␮L
cell
cul u e
medium
con aining
he
co esponding
subs a e
(Table
1).
A e
incu-
ba ion
a
37 ◦C,
fluo escence
(FLx800,
BioTek,
Po on,
UK)
o
abso bance
(Infini e
M200,
Tecan,
Männedo ,
Swi ze land)
was
measu ed.
S anda d
cu es
we e
ob ained
by
seeding
a
Table
1
–
Cy ocompa ibili y
assay
pa ame e s.
Subs a e
Incuba ion
ime
(min)
Measu emen
Resazu in,
10%
90
Fluo escence
–
exci a ion:
560
nm,
emission:
590
nm
WST-8,
10%
80
Abso bance
–
460
nm
( e e ence:
650
nm)
MTS,
20% 60
Abso bance
–
490
nm
( e e ence:
630
nm)
ange
o
cell
concen a ions
in
96
well
pla es,
2
h
p io
o
each
assay.
2.2.5.
S a is ical
analyses
One-way
analysis
o
a iance
(ANOVA)
and
pos -hoc
Tukey’s
es s
we e
used
o
de e mine
significance
(p
≤
0.05)
be ween
composi e
o mula ions
(UP,
UT,
BP,
BT
and
Z250).
Two-way
ANOVA
and
pos -hoc
Tukey’s
es s
we e
also
used
in
o de
o
de e mine
he
significance
o
he
e ec s
o
mul iple
ac-
o s,
e.g.
UDMA
s.
Bis-GMA
and
PPGDMA
s.
TEGDMA.
Fo
all
echniques,
he
s anda d
de ia ion
(SD)
o
each
o mula ion
was
displayed
on
g aphs,
excep
in
he
case
o
polyme iza ion
sh inkage,
whe e
he
SD
was
a e aged
o
each
echnique,
due
o
he
high
a iabili y
o
he
olume
change
me hod.
S a is-
ical
significance
is
p esen ed
on
g aphs
(*p
<
0.05,
**p
<
0.01,
***p
<
0.005
o
****p
<
0.001).
3.
Resul s
3.1.
Handling
p ope ies
and
we -poin
All
expe imen al
o mula ions
had
we -poin s
o
33.3
±
0.4
ol%
liquid.
A
his
le el,
o mula ion
pas es
con aining
Bis-GMA
el
conside ably
less
malleable
du ing
specimen
moulding
han
hose
p oduced
wi h
UDMA.
A
40
ol%
liquid,
all
o mula ions
we e
sligh ly
mo e
malleable,
bu
o mula ions
con aining
Bis-GMA
s ill
exhibi ed
lowe
malleabili y
han
hose
con aining
UDMA.
Z250
had
handling
p ope ies
be ween
hose
o
Bis-GMA-
and
UDMA-con aining
composi es.
No
disce nible
di e ence
in
handling
p ope ies
was
obse ed
be ween
PPGDMA
and
TEGDMA.
3.2.
Polyme iza ion
p ope ies
3.2.1.
Con e sion
Expe imen al
composi es
con aining
UDMA
had,
on
a e age,
1.2
and
1.3
imes
highe
con e sion
a
1
and
4
mm
dep h,
espec i ely,
han
hose
con aining
Bis-GMA
(a e age
p- alue
o
bo h
dep hs
=
0.001)
(Fig.
1a).
Replacemen
o
TEGDMA
wi h
PPGDMA
u he
inc eased
con e sion
by
1.1
and
1.2
imes
a
1
and
4
mm,
espec i ely
(a e age
p- alue
o
bo h
dep hs
<
0.01).
UP
had
he
highes
con e sion
(68%
and
65%
a
1
and
4
mm).
A
bo h
dep hs,
UP’s
high
con e sion
was
highly
s a is ically
sig-
nifican
compa ed
o
BP,
BT
and
Z250
(p
<
0.001)
and
a
1
mm,
i
was
significan ly
highe
han
ha
o
UT
(p
<
0.05).
Z250
had
he
lowes
(50%
and
39%
a
1
and
4
mm)
and
was
compa a-
ble
o
BT.
Composi es
con aining
Bis-GMA
(BP,
BT
and
Z250),
showed
a
significan
educ ion
in
con e sion
upon
inc easing
dep h
om
1
o
4
mm.
268
d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
Fig.
1
–
(a)
Con e sion
o
composi es
a
1
and
4
mm
dep h.
Columns
ep esen
mean,
e o
ba s
ep esen
SD.
(b)
Sh inkage
o
1
mm
hick
composi e
discs,
calcula ed
om
con e sion
o
olume
change.
Columns
ep esen
mean,
e o
ba s
ep esen
mean
SD
o
he
co esponding
echnique
(±0.05
and
±0.23
ol%
o
con e sion
and
olume
change,
espec i ely).
(c)
Dep h
o
cu e
o
composi es.
(d)
Wa e
so p ion
o
composi es
a e
imme sion
in
deionized
wa e
o
one
week.
(c,
d)
C osses
ep esen
mean,
boxes
ep esen
25–75
pe cen iles,
e o
ba s
ep esen
SD.
3.2.2.
Sh inkage
Based
on
con e sion
a
1
mm
dep h,
he
sh inkage
o
UDMA-
con aining
composi es
was
p edic ed
o
be
1.2
imes
highe ,
on
a e age,
han
hose
con aining
Bis-GMA
(p
<
2
×
10−5)
(Fig.
1b).
Composi es
con aining
TEGDMA
we e
es ima ed
o
ha e
1.1
imes
highe
sh inkage
han
hose
con aining
PPGDMA
(p
<
0.04).
When
sh inkage
was
de e mined
by
olume
change,
howe e ,
no
s a is ically
significan
e ec
o
monome
could
be
obse ed.
This
was
due
o
he
high
s anda d
de ia ion
o
he
echnique.
The
sh inkage
o
Z250
could
no
be
calcula ed
using
con e sion
da a,
since
i s
exac
composi ion
was
unknown.
The
measu ed
sh inkage,
howe e ,
o
Z250
was
compa able
o
all
expe imen al
o mula ions.
3.2.3.
Dep h
o
cu e
The
dep h
o
cu e
(Fig.
1c)
was
highly
compa able
o
all
com-
posi es
(2.7
±
0.25
mm),
ega dless
o
monome
composi ion
(F
=
0.96).

d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
269
3.2.4.
Wa e
so p ion
The
wa e
so p ion
o
o mula ions
con aining
TEGDMA
was,
on
a e age,
1.2
imes
highe
han
ha
o
hose
con aining
PPGDMA
(p
<
0.02)
(Fig.
1d).
On
a e age,
UDMA
induced
∼1.1
imes
highe
wa e
so p ion
han
Bis-GMA,
al hough
his
was
no
s a is ically
significan .
Z250
had
he
lowes
wa e
so p-
ion,
compa able
o
ha
o
BP.
UT
had
he
highes
wa e
so p-
ion,
significan ly
highe
han
ha
o
BP
and
Z250
(p
<
0.05).
3.3.
Mechanical
p ope ies
Expe imen al
composi es
con aining
UDMA
had
significan ly
highe
s eng h
(153–158
MPa)
han
hose
con aining
Bis-GMA
Fig.
2
–
(a)
Biaxial
flexu al
s eng h
and
(b)
modulus
o
ensile
elas ici y
o
composi es
a e
s o age
in
deionized
wa e
a
37 ◦C
o
one
week.
C osses
ep esen
mean,
boxes
ep esen
25–75
pe cen iles,
e o
ba s
ep esen
SD.
(p
=
1
×
10−7).
The
s eng h
o
BP
was
significan ly
highe
han
ha
o
BT
(127
and
99
MPa,
espec i ely;
p
<
0.05)
(Fig.
2a).
The
modulus
o
expe imen al
o mula ions
con aining
PPGDMA
was,
on
a e age,
sligh ly
lowe
han
ha
o
o mula ions
con-
aining
TEGDMA
(6.15
and
6.75
GPa,
espec i ely;
p
=
0.009)
(Fig.
2b).
Z250
had
he
highes
s eng h
(173
MPa)
and
lowes
modulus
(5.3
GPa).
3.4.
Cy ocompa ibili y
3.4.1.
Compa ison
o
esazu in,
WST-8
and
MTS
assays
The
appa en
cell
densi y
o
HGF
a e
48
and
72
h
cul u e
in
se um- ee
DMEM
is
shown
in
Fig.
3.
Resazu in
and
WST-8
assays
epo ed
a
simila
inc ease
in
appa en
cell
densi y
om
30,000
o
∼49,000
cells/cm2be ween
0
and
48
h.
MTS
epo ed
a
lesse
inc ease
in
appa en
cell
densi y
( om
30,000
o
37,000
cells/cm2a e
48
h).
A e
72
h
in
cul u e,
he
esazu in
assay
epo ed
cell
densi y
o
ha e
inc eased
o
113,000
cells/cm2,
whe eas
he
WST-8
assay
epo ed
cell
densi y
o
be
74,000
cells/cm2.
MTS
again
epo ed
only
a
small
inc ease
in
appa en
cell
densi y
(45,000
cells/cm2).
Since
he
assays
epo ed
di e en
cell
densi ies
in
he
con ols
a
di e en
ime-poin s,
subsequen
da a
a e
no -
malized
o
he
con ol
o
he
co esponding
assay
and
ime-poin
and
a e
epo ed
in
e ms
o
ela i e
me abolic
ac i i y.
3.4.2.
Composi e
componen
cy ocompa ibili y
The
ela i e
me abolic
ac i i y
o
su i ing
HGF
ollowing
exposu e
o
se ial
dilu ions
o
liquid
phase
componen s,
as
well
as
ollowing
a
subsequen
eco e y
pe iod,
is
p esen ed
in
Fig.
4.
The
dashed
lines
ep esen
he
mean
alue
ob ained
o
he
DMEM
con ol
o
each
co esponding
ime-poin
and
assay.
All
componen s
caused
a
ypical,
concen a ion-
dependen
educ ion
in
cell
numbe
as
concen a ion
was
Fig.
3
–
Ini ial
seeding
densi y
o
HGF
de e mined
by
cell
coun ing
(0
h,
c oss),
and
appa en
densi y
a e
cul u e
in
DMEM
o
48
h
(unfilled
symbols)
and
72
h
(filled
symbols),
as
assessed
by
esazu in
(ci cle),
WST-8
( iangle)
and
MTS
(squa e)
assays.
E o
ba s
ep esen
SD.
270
d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
Fig.
4
–
Me abolic
ac i i y
( ela i e
o
he
co esponding
con ol)
o
su i ing
HGF
a e
cul u e
in
aqueous
solu ions
o
composi e
liquid
phase
componen s
(a)
CQ,
(b)
DMAEMA,
(c)
UDMA,
(d)
Bis-GMA,
(e)
PPGDMA
o
( )
TEGDMA.
Resazu in
(ci cle),
WST-8
( iangle)
and
MTS
(squa e)
assays
we e
pe o med
a e
24
h
cul u e
in
he
solu ions
(48
h
ime-poin ,
unfilled
symbols),
and
a e
a
subsequen
24
h
eco e y
pe iod
in
DMEM
(72
h
ime-poin ,
filled
symbols).
Componen
concen a ions
anged
om
0.001
o
10
mM
(a
and
d)
o
0.01
o
100
mM
(b,
c,
e
and
).
Dashed
lines
ep esen
he
mean
alue
o
he
con ol,
e o
ba s
ep esen
SD.
d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
271
inc eased,
hough
he
ex en
o
his
ela ionship
depended
on
he
assay.
In
he
case
o
all
componen s,
a
concen a ion
o
10
mM
was
su ficien
o
cause
close
o
100%
educ ion
in
me abolic
ac i i y.
A e
48
h,
WST-8
indica ed
a
di ec
co ela ion
be ween
CQ
concen a ion
(Fig.
4a)
and
me abolic
ac i i y,
wi h
0.001
and
0.01
mM
ha ing
no
significan
e ec
on
me abolic
ac i i y.
As
concen a ion
was
inc eased
o
0.1,
1
and
10
mM,
me abolic
ac i i y
was
educed
by
17,
33
and
98%,
espec i ely.
Resazu in
epo ed
simila
le els
(±5%)
o
me abolic
ac i i y
a
mos
con-
cen a ions,
whe eas
MTS
alues
we e
up
o
15%
highe .
A e
72
h,
esazu in
epo ed
simila
le els
o
ela i e
me abolic
ac i i y
o
a e
48
h.
WST-8,
howe e ,
indica ed
simila
le els
o
me abolic
ac i i y
o
he
con ol
(±7%)
a
all
concen a ions
excep
≥10
mM,
a
which
cells
did
no
eco e .
MTS
showed
ele a ed
me abolic
ac i i y
(10–38%
g ea e
han
ha
o
he
con ol)
a
all
concen a ions
a e
72
h,
excep
≥10
mM.
By
con as ,
a
48
h,
0.01
mM
DMAEMA
(Fig.
4b)
caused
an
86–95%
educ ion
in
me abolic
ac i i y
in
all
assays,
wi h
no
eco e y
a e
72
h.
All
assays
indica ed
ha
concen a ions
≥0.1
mM
caused
o al
inhibi ion
o
me abolic
ac i i y.
Simi-
la ly,
a
a
c i ical
concen a ion
o
0.01
mM
o
highe ,
UDMA
(Fig.
4c)
and
TEGDMA
(Fig.
4 )
bo h
inhibi ed
me abolic
ac i i y
in
all
assays.
Bis-GMA
(Fig.
4d)
appea ed
o
be
mo e
cy ocompa ible
han
DMAEMA,
UDMA
and
TEGDMA.
A e
48
h,
WST-8
indi-
ca ed
ha
concen a ions
o
0.001–0.01
mM
had
no
e ec
on
me abolic
ac i i y.
I
ell,
howe e ,
by
7
and
66%
upon
inc eas-
ing
concen a ion
o
0.1
and
1
mM,
espec i ely.
The
o he
assays
showed
a
simila
end,
bu
a
0.001–0.1
mM
he
alues
epo ed
by
esazu in
and
MTS
we e
∼10–15%
and
∼23–28%
lowe ,
espec i ely.
A e
72
h,
alues
ob ained
by
WST-8
assay
we e
ele a ed
by
be ween
7
and
20%
compa ed
o
he
p e i-
ous
ime-poin ,
excep
a
10
mM.
A
0.001–0.1
mM,
me abolic
ac i i y
appea ed
highe
han
he
con ols.
Resazu in
epo ed
simila
alues
(±10%)
a e
72–48
h.
HGF
exposed
o
PPGDMA
(Fig.
4e)
exhibi ed
mo e
com-
plex
ends.
The
h ee
assays
ga e
di e en
esul s
o
concen a ion-dependen
e ec s
on
ela i e
me abolic
ac i -
i y.
Acco ding
o
all
assays,
a
≥10
mM,
PPGDMA
comple ely
inhibi ed
me abolic
ac i i y
a e
48
h
and
his
did
no
eco e
a e
72
h.
Upon
lowe ing
concen a ion,
he e
was
a
small
inc ease
in
ac i i y
acco ding
o
he
WST-8
and
MTS
assays,
bu
i
emained
well
below
con ol
alues
and
was
no
enhanced
by
p o iding
addi ional
eco e y
ime.
Con-
e sely,
a
48
h
and
PPGDMA
le el
below
0.1
mM,
he
cell
ac i i y
acco ding
o
he
Resazu in
es
ended
o
highe
al-
ues
han
wi h
he
con ol.
By
72
h,
howe e ,
his
“excess”
me abolic
ac i i y
was
down
o
con ol
le els.
Concen a-
ions
o
0.01,
0.1
and
1
mM,
PPGDMA
caused
a
educ ion
in
WST-8
ac i i y
o
76,
85
and
92%,
espec i ely,
a e
48
h.
These
le els
emained
unchanged
a e
72
h.
MTS
showed
simila
alues
o
WST-8.
By
con as ,
a e
48
h,
esazu in
ac i i y
was
ele a ed
by
63
and
35%
abo e
he
con ol
a
0.01
and
0.1
mM,
espec i ely,
and
a
1
mM
was
simila
o
he
con ol.
A e
72
h,
hese
alues
ell,
wi h
esazu in
ac i i y
emaining
12%
abo e
he
con ol
a
0.01
mM,
and
being
7
and
80%
lowe
han
he
con ol
a
0.1
and
1
mM,
espec i ely.
3.4.3.
Composi e
ex ac
cy ocompa ibili y
The
me abolic
ac i i y
o
HGF
a e
cul u e
in
composi e
ex ac s
is
p esen ed
in
Fig.
5.
Significan
di e ences
we e
again
obse ed
be ween
di e en
assays.
In
o de
o
acili a e
elucida ion
o
ends,
da a
a e
exp essed
as
a
pe cen age
o
he
con ol
o
each
co esponding
ex ac
(1
o
7
days)
and
assay
ime-poin
(48
o
72
h).
A e
24
h
exposu e
o
1
day
composi e
ex ac s
(48
h
ime-
poin ),
a
gene al
downwa d
end
in
esazu in
me abolism
was
obse ed
as
ollows:
UP
>
UT
≥
BP
>
BT
≥
Z250.
Al hough
WST-8
ac i i y
di e ed
sligh ly
(UP
≈
UT
>
BP
≈
BT
>
Z250),
bo h
assays
confi med
ha
he
ex ac s
om
he
lowe
quad an
o
4
mm
deep
composi e
samples
con aining
UDMA
had
less
e ec
on
he
48
h
me abolic
ac i i y
han
hose
con aining
Bis-
GMA.
In
he
case
o
esazu in,
his
was
s a is ically
significan
(p
=
0.001).
They
also
showed
ha
Z250
ex ac s
we e
less
cy o-
compa ible
han
all
o
he
expe imen al
o mula ions.
MTS
showed
no
significan
di e ences
be ween
expe imen al
o -
mula ions
bu
confi med
he
low
cy ocompa ibili y
o
Z250.
Me abolic
ac i i y
in
all
assays
a ied
om
being
simila
o
he
con ol
o
UP,
down
o
∼50%
o
con ol
o
Z250.
Composi es
con aining
TEGDMA
caused
a
sligh
bu
significan
educ ion
in
esazu in
ac i i y
compa ed
o
PPGDMA
(p
<
0.01),
bu
WST-
8
and
MTS
assays
showed
no
clea
ends
wi h
ega ds
o
he
e ec
o
diluen
monome .
A e
72
h,
simila
ends
we e
obse ed
in
all
h ee
assays,
wi h
me abolic
ac i i y
alling
o
app oxima ely
50%
o
ha
o
he
con ol
o
all
expe imen al
ma e ials.
The
only
di -
e ences
be ween
assays
we e
obse ed
wi h
Z250,
whe e
esazu in
ac i i y
had
emained
simila
o
he
p e ious
ime-
poin
a
∼50%,
bu
WST-8
ac i i y
had
allen
om
53
o
37%
and
MTS
ac i i y
had
allen
om
53
o
33%.
These
di e ences,
howe e ,
we e
no
s a is ically
significan .
Resazu in
and
WST-8
sugges ed
ha
7-day
ex ac s
we e
mo e
cy ocompa ible
han
1
day
ex ac s,
since
me abolic
ac i i y
e u ned
o
con ol
le els
a e
72
h.
The
end
o
he
48
h
esazu in
assay
a e
7
days
ex ac ion
was
simila
o
he
co esponding
1
day
ex ac ,
bu
wi h
sligh ly
highe
alues.
Fo
example,
48
h
assays
showed
ha
BT
–
he
leas
cy ocom-
pa ible
expe imen al
composi e
–
had
75%
esazu in
ac i i y
ela i e
o
he
con ol
a e
7
days
ex ac ion,
compa ed
o
56%
a e
1
day.
In
he
case
o
all
expe imen al
o mula ions,
WST-8
ac i i y
was
simila
o
he
con ol
o
sligh ly
ele a ed
by
up
o
11%
a e
48
h.
The
7-day
ex ac
o
Z250,
howe e ,
s ill
caused
a
significan
educ ion
in
WST-8
ac i i y.
A e
72
h,
he
esazu in
and
WST-8
ac i i y
o
HGF
exposed
o
7-day
ex ac s
had,
in
gene al,
eco e ed.
Resazu in
ac i i y
o
BT
and
Z250
emained
sligh ly
educed
a
89
and
82%,
espec i ely.
A e
72
h,
WST-8
ac i i y
o
HGF
exposed
o
all
composi e
ex ac s
was
ele a ed
abo e
ha
o
he
con ol
by
be ween
10
and
31%.
The
MTS
assay
ga e
significan ly
di e en
alues
com-
pa ed
o
he
o he
wo
assays
o
he
7-day
ex ac s.
A e
48
h,
MTS
ac i i y
o
HGF
exposed
o
all
expe imen al
o -
mula ions
was
60–75%
highe
han
he
con ol
and
emained
simila
a e
72
h.
Z250
alues
we e
simila
o
hose
epo ed
by
WST-8.
UDMA
was
also
shown
o
be
mo e
cy ocompa ible
han
Bis-GMA
in
7-day
ex ac s
(48
h
esazu in
assay,
p
=
0.03;
72
h
WST-8
assay,
p
=
0.0008;
72
h
MTS
assay,
p
=
0.007).
WST-8
ac i i y
was
also
significan ly
highe
in
cells
exposed
o
7-day
272
d
e
n
a
l
m
a
e
i
a
l
s
3
2
(
2
0
1
6
)
264–277
Fig.
5
–
Me abolic
ac i i y
( ela i e
o
he
co esponding
con ol)
o
HGF
a e
48
(a
and
c)
o
72
(b
and
d)
h
cul u e
in
1
(a
and
b)
o
7
(c
and
d)
day
composi e
ex ac s,
assessed
by
esazu in
(da k
g ay),
WST-8
(black)
and
MTS
(ligh
g ay)
assays.
Dashed
lines
ep esen
he
mean
alue
o
he
con ol,
e o
ba s
ep esen
SD.
ex ac s
o
PPGDMA-con aining
composi es
han
TEGDMA-
con aining
composi es
a e
48
h
(p
=
0.04).
4.
Discussion
As
hypo hesized,
den al
composi es
con aining
UDMA
had
significan ly
highe
monome
con e sion
and
sligh ly
be e
cy ocompa ibili y
han
hose
con aining
Bis-GMA.
Simila ly,
composi es
con aining
PPGDMA
achie ed
mo e
comple e
con-
e sion
han
hose
con aining
TEGDMA.
Impo an ly,
he
use
o
UDMA
and/o
PPGDMA
as
he
sole
bulk/diluen
monome
had
no
de imen al
e ec
on
he
sh inkage
o
dep h
o
cu e
o
he
ma e ials.
In
addi ion,
composi es
con aining
UDMA
had
imp o ed
handling
p ope ies
compa ed
o
hose
con aining
Bis-GMA.
The
a ia ion
in
he
con e sion
o
he
composi es
is
a ibu ed
o
di e ences
in
he
chemical
s uc u es
(Fig.
6)
and
physical
cha ac e is ics
(Table
2)
o
hei
cons i uen
monome s.
Al hough
UDMA
is
a
sligh ly
smalle
molecule
han
Bis-GMA
and,
as
a
esul ,
has
sligh ly
highe
double
bond
concen a ion
( a io
o
double
bonds
pe
molecule
o
molecula
mass),
i s
lowe
glass
ansi ion
empe a u e
(Tg)
and
g ea e
flexibili y
enable
mo e
comple e
c oss-linking
[15].
The
bulky
a oma ic
g oups
o
Bis-GMA
cause
s e ic
hind ance,
eflec ed
in
i s
e y
high
iscosi y
alue,
which
is
∼80
imes
ha
o
UDMA.
This
educed
flexibili y
limi s
he
likelihood
o
me hac yla e
g oups
coming
in o
con ac
wi h
each
o he
and
binding.
PPGDMA
has
mo e
han
wice
he
molecula
mass
o
TEGDMA
and
as
a
esul ,
i s
g ea e
flexibili y
and
lowe
double
bond
concen a ion
esul
in
imp o ed
con e sion.
The
simila
dep h
o
cu e
o
he
composi es
is
likely
o
be
due
o
he
simila
e ac i e
indices
o
he
monome s
used