The Indonesian J Dent Res, 2011, Volume 1, No.

2
Published online: http://the-indonesian-jdr.fkg.ugm.ac.id

The Effect of Calcium Hydroxide on

Fibroblast Cells Viability

Vincentia Adya Paramitta

Tetiana Haniastuti
Heni Susilowati

Department of Oral Biology, Faculty of Dentistry,

Universitas Gadjah Mada, Yogyakarta, Indonesia

E-mail: drgheni.susilowati@gmail.com
Received August 15, 2010; Accepted December 10, 2010

Abstract
Calcium hydroxide [Ca(OH)2] is widely used as medicament in dental pulp and root canal therapy. Previous
studies demonstrate the ability of calcium hydroxide to induce necrosis in dental pulp tissue. However, the
mechanism of tissue destruction remains unknown. The aim of this study was to investigate fibroblast cell
viability in response to calcium hydroxide exposure. In this study, Vero fibroblast cell line was treated with
various concentrations of calcium hydroxide for 24 hours. Cell viability was measured by using MTT assay.
Our results showed significant decrease in cell viability after exposed with calcium hydroxide at concentration
62.5 and 125 µg/ml. The result indicated that calcium hydroxide induced cell death in Vero cell line in a dose-
dependent manner. This study suggests that fibroblast cell death may involved in the mechanism of pulp
tissue necrosis caused by calcium hydroxide.

Keywords: Calcium hydroxide, cell viability.

Introduction used in many dental treatments such as pulp capping,

Dental pulp is a connective tissue that is
uniquely situated within the rigid encasement of
mineralizeddentin1. Fibroblast is the most distributed
cell in the dental pulp tissue and widely distributed
in cell-rich zone. Fibroblast main function is to
synthesize colagen tipe I and III, which take part in
tissue remodelling1,2.
Endodontic treatment is a procedure to maintain
the health of the pulp and the periapical tissue3.
Calcium hydroxide [Ca(OH)2] has been widely used as
medicament in endodontic treatment since Hermann
used it for the first time in 19204. This medicament is
pulpotomy, apexification, root perforation, and internal
or external root resorption5-11. Calcium hydroxide
has antimicrobial effect5that further more can give
therapeutic effect and it has the ability to induce
reparative dentin and dentinal bridge formation12.
Previous in vivo study demonstrated the potencial
of calcium hydroxide in inducing dental pulp tissue
destructions13. However the underlying mechanism
of tissue destruction is not well understood. As an
initial step to clarify the molecular mechanism of
Ca(OH)2-induced pulp tissue destruction, this study
was subjected to investigate fibroblasts cell viability
after Ca(OH)2 exposure in vitro.

105
 Paramittaet al.

Materials and Methods Paramittaet significant

al. difference was found between the
Paramittaet Paramittaet al. al.
group treated with calcium hydroxide 125 µg/ml
Paramitta et al.
InMaterials
this study, and VeroMethods fibroblast cells were culturedsignificant and 250 µg/ml.
significant difference was found found between the
Materials
Materials and Methods
and Methods difference
significant difference was found was between between the the
and maintained in M199 culture medium (Gibco,group group group
treated treated
with
treated with
calcium
with the calcium
hydroxide
calcium hydroxide125 with 125
µg/ml µg/ml
Materials and Methods was found between grouphydroxide
treated 125calciumµg/ml
Paisley,
Instudy, UK)
this study,
study, suplemented
Vero fibroblast
fibroblast with 10%
cells were fetal
were cultured bovine
cultured and 250and
and 250
µg/ml.
250 µg/ml.
µg/ml.
In thisIn this Vero fibroblast
Vero cells were cells cultured hydroxide 125 μg/ml and 250 μg/ml.
serum (Gibco), 2%
and maintained
maintained in M199 penicillin
M199 culture andmedium streptomycin (Gibco,
and maintained
and in M199 in cultureculture medium medium (Gibco,(Gibco,
In this study,
(Gibco),
Paisley, andUK)0.5% Vero fibroblast with
fungizone
suplemented cells were
(Gibco). 10% The cultured
confluent
fetal and
bovine 120
Paisley, UK) suplemented
Paisley, UK) suplemented with 10% with fetal 10% bovine
fetal bovine
maintained
culture
serum was
(Gibco),in M199
harvested 2% culture and
penicillin medium
plated and (Gibco,
into 96 Paisley,
streptomycin wells
serumserum (Gibco), 2% penicillin
(Gibco), 2% penicillin and streptomycin
and ostreptomycin
UK)
plate
(Gibco), suplemented
for and overnight with
0.5% fungizone incubation
fungizone 10%(Gibco). fetal
at 36bovine C. The
The serum
first
confluent 100
(Gibco), and 0.5%
(Gibco), and fungizone
0.5% 4 (Gibco). The
(Gibco). confluent
The confluent 120 120 120
(Gibco),
group
culture of 2%
cells(2 penicillin
was harvested x 10
harvested and
cells/well) streptomycin
and plated platedwas then
into (Gibco),
treated
96 wells
wells
cultureculture
was harvested and plated into 96 wells

(%)
was and into 96 80
and
with
plate 0.5%
various fungizone
for overnightconcentrations
overnight (Gibco).
incubation The
of atconfluent
calcium 36ooC. culture
C.hydroxide
The first
plate plate
for overnight
for incubation incubation at 36oat C. 36The first
The first 100 100 100

Cell Viability
group(62.5 ofµg/ml, 125 µg/ml, and 250
was
group harvested cells(2 and plated
44 cells/well) into 96 wellsthenplate for
of cells(2
group of cells(2 xx 10
x 104 cells/well) 10 cells/well) was then was
was treated
then treated
treated 60

(%)
Cell Viability (%)

(%)
µg/ml),whereasanother
overnight
with
with various various incubation
concentrationsconcentrations at one
36 o
C.
of calcium was
The
of left
first
calcium untreated
group
hydroxide of
hydroxide as
cells 80 8080
with various concentrations of calcium hydroxide

Viability
(62.5 a(62.5
(2 negative
xµg/ml, control.Each
10 cells/well) 125 was concentration
thenµg/ml, treated
and with of calcium
various

CellViability
(62.5
4
µg/ml, 125 and 250 40
µg/ml, 125 µg/ml, µg/ml, and 250 250 60 6060
hydroxide
concentrations was
µg/ml),whereasanother
µg/ml),whereasanother addedof to
one was the
calcium one cells
left and
hydroxide
was left
untreated incubated (62.5μg/
untreatedas for as
µg/ml),whereasanother one was left untreated as 20
72 hours in control.Each
5%and , 37oconcentration
COconcentration C. Allwhere treatment was
aml,
a negative 125μg/ml,
a negative
negative
control.Each control.Each 2250μg/ml),
concentration of as
calcium
of another
of calcium
calcium 40 4040

Cell
performed
one
hydroxide
hydroxide was
hydroxide
was added in
left
was 5
was added replications.
untreated
added
to theto to
cellsasthe
theanda Cell
cells viability
negative and
cellsincubated
and incubated was
control.
incubated
for forthen
Eachfor 0
measured
concentration
72 hours
hours by
inCO using
of
5% CO o MTT
calcium , 37 oassay.
ohydroxide
C. All Thewas was
treatment following
addedwas wasto 20 2020
72 hours
72 in 5%in 5% 2, 37 CO 2C.
,
2 37 All C. treatment
All treatment
equation
the
performed cells
performed
performed in 5and was used
inincubated to find
55 replications.
replications.
in replications. for 72
Cell the percentage
hours
Cell
viability
Cell in
viability
was
viability 5%then CO of2,Vero
was
was 37
then
o
then C.
fibroblast
All treatment cells viability
was
13
performed . in 5 replications. Cell
0 00
measured measured
measured by using by
by usingusing
MTT assay. MTT
MTT assay. assay.
The following The following
The following
equationviability
equationwas was
equation usedwas
was thenused
to
used measured
find totothe find
find by
theusing
percentage
the percentage
percentage MTT of Veroassay. The
of Vero
of Vero
13
following
fibroblast
fibroblast equation
cellsviability
cells viability
fibroblast cells . was used
viability
13 13
.. OD into find the percentage
treated group
of Vero fibroblast Viabilitycells (%)= viability 13
. ×100%(1) Figure 1. Calcium hydroxide induced cell death in
OD in nega�ve control group
Vero fibroblast cells. The untreated cells showed
OD in treated ODinin treatedgroup
group group almost1.hundred
Figure Calcium hydroxide percent living induced cells.cellThe death treatment
in Vero
OD = opticalViability density
Viability
(%)= OD(%)=
Viability
OD treated
(%)= OD in nega�ve control ×100% ×100%
(1)
×100% (1)
(1) of various concentrations of Ca(OH) caused cell
in nega�ve
OD in control
nega�vegroup
control groupgroup fibroblast cells. The untreated cells showed2 almost
death inpercent
hundred dose-dependent living cells.manner (p<0.05).
The treatment ofThe data
various
Figure
1.
Figure 1. Calcium
Figureconcentrations
were Calcium
presented
Calcium
1. hydroxide hydroxide
of hydroxide
Ca(OH)
as means 2 caused
induced induced
±cell cell
SDdeath
induced cell
ofcell death death
representative
in
deathVeroininindose-
Vero
Vero
OD==optical
OD = optical
OD optical
densitydensitydensity
To determine whether the treatment of calciumfibroblast fibroblast
dependent
cells. The
fibroblast
experiments. cells.untreated
manner
cells. The
The untreated
(p0.05).
untreatedcells cells were
Theshowed
data
cells showed
showed presented
almost almost
almost
hundred
ashundred
percent
means
hundred percent
living
±percent
SD livingThe
cells.
of representative
living cells.
cells. The
treatment
The treatment
experiments.
treatmentof various of various
of various
hydroxide had significant effect on the cell viability,concentrations concentrations
of Ca(OH) of Ca(OH)
Ca(OH) caused cell deathdeath in dose-
dose-
concentrations of 2 caused cell death
22 caused cell in dose- in
the To
To determine To
data determine
were
determine
To determine whether whether
analyzed
whether
whether by
the treatment the
using
the treatment
one-way
treatment
the treatment of calcium of
of calcium
analysis
calcium
of calcium dependent
dependent Figure
manner
dependent manner
1 and
(p0.05).
manner (p0.05).
Table 1
The
(p0.05). data The
demonstrate
The data
were
data were
that
presented
were presented
the
presentedgroup
hydroxide had significant effect on the cell viability, as means
as Figure
asmeans
means
± SD of 1SDSDand
±±representative
of Table experiments.
ofrepresentative
representative 1 demonstrate
experiments.that the
experiments.
of
hydroxide variance
hydroxide
hydroxide (ANOVA).
had
had significant
had significant
significant The effect
effect Post-Hoc
effect
on theon oncellleast
the
the significant
cell
viability,
cell viability,
viability, treated with the largest concentration of calcium
the data were analyzed by using one-way analysis group treated with the largest concentration of
difference
the data the data
were (LSD)
were
analyzed test
analyzed
by was
using
the data were analyzed by using one-way analysis by doneusing
one-way to determine
one-way analysis the
analysis hydroxide (250 µg/ml) has the lowest percentage
significant Figure calcium
Figure
1 and hydroxide
1 Table
and Table 1(250
Table 11µg/ml)
demonstrate has
demonstrate that thethe lowest
that the
of
of variance
of variance
significance
of variance
variance
(ANOVA). (ANOVA).
of difference
(ANOVA).
(ANOVA).The Post-Hoc Theamong
The Post-Hoc
Post-Hoc
Post-Hocleastgroups. least
least significant
significant
least of Figure
viable 1cells.
and The increasing demonstrateconcentration that the
of
difference (LSD) test was done to determine thegroup percentage
group
treated
group treated
with
treated of viable
with
the the
largest
with theadded cells.
largest The
concentration
concentration
largest concentration increasing
of of
of
difference
difference
difference (LSD) (LSD) (LSD)was
test testdone
test was done
was done
to to determine
determine
to determine the the the calcium hydroxide to the cells caused
Results and Discussion concentration
calcium calcium
hydroxide
calcium hydroxide
hydroxide of the
(250 (250 calcium
(250
µg/ml)µg/ml) µg/ml)hydroxide
has the has added
the
lowest to
lowest
significance
significance
significance
significance of differenceof
of difference
ofdifference
difference
amongamong among
among
groups. groups.
groups.
groups. decrease
the cellsofin causedtheofpercentage of viablehas Vero the lowest
cells.
percentage
percentage
percentage of decrease
viable viable
viablecells. cells. in
ThetheThe
cells. percentage
The
increasing increasing
increasing of
viable There
Vero
concentration are
cells.many
of the typescalcium of preparation
hydroxide ofadded
calcium to
Results
TheResults
Results and and
resultDiscussion
obtained
and Discussion
Discussion in this study is presented in concentration of the calcium
concentration of the calcium hydroxide added to
hydroxide used in dentistry.
hydroxide
The
added
typespercentage
are
to
available
Figure 1 and Table 1. The results of ANOVAthe cells theThere
cells
the caused are
cells caused many
caused
decrease types
decrease
decrease of preparation
in
in theinpercentage the
the14percentage of calcium
of of
of
as points, setting, and non-setting . Pure calcium
The result
demonstrated
The result
The result
obtained obtained
a in
obtained significant
thisin instudythis study
decrease
is isisinpresented
presented presented
cell viability
in viablehydroxide
in viable
viable Vero
Vero cells.
Vero used in dentistry. The types are available
cells.
cells.
The result obtained in thisthis study study
is presented in Figure in hydroxide
as There
points, preparation
are many types wasofused in14of.this
preparation study.
ofcalcium The
calcium
Figure
Figureafter1 and
Figure 11 Table
exposed and with
and Table
1. The
Table 1. results
calcium
1. Thehydroxide
The results
of ANOVA
results of(p<0.05),
of ANOVA
ANOVA There There are manyand
are many setting,types of
types non-setting
preparation
of preparation Pure
calciumof calcium
1 and Table 1. The results of ANOVA demonstrated result
hydroxide
hydroxide of this usedstudy showed
in dentistry.
preparationwas dentistry. that
Thein
used Ca(OH)
typesthis might
2are
study. have
available
The
demonstrated
indicating
demonstrated
demonstrated athat aa significant
significant
calcium
significant decrease
hydroxidemight
decrease in cellin
decrease in have
viability
cell abilityhydroxide
cell viability
viability used in
hydroxide used dentistry.
in The types The are
types available
are available
aafter
significant decrease in cell viability after exposed ability
as points,
result to
points,
of thisinduce
setting,
study fibroblast
and cell
non-setting
showed 14
that death.14
. PureCa(OH)
14
..calciumThe
Pure2calcium result
calcium
might
after to
afterinduce
exposed exposed
exposedcell
withdeath. withIncalcium
calcium
with calcium
addition,
hydroxide hydroxide
Post-Hoc
hydroxide (p<0.05), LSD testas points,
(p<0.05),
(p<0.05), as setting, and
setting, non-setting
and non-setting Pure
with
indicatingcalcium that hydroxide
calcium (p<0.05), indicating
hydroxidemight have that
ability revealed
hydroxide
have that calcium
preparationwas hydroxide
hydroxide preparationwas used in this study. The
hydroxide ability
preparationwas to induce used used
fibroblast
in this atin concentration
cellthis
study. study.
death.
The Theof
The
showed
indicating thatacalcium
indicating significant
that calcium difference
hydroxidemight hydroxidemight (p<0.05)
have ability havebetweenability
calcium hydroxide might have ability to LSD induce 62.5
result
resultresult µg/ml
of this or higher
ofrevealed
this
study study
showed might
showed
that that cause that
calcium
Ca(OH) cellCa(OH)death
Ca(OH) inmight
hydroxide Vero
to induce
toto induce
control induce
cellgroup cell death.
death.
cell death.
with allIn
In addition, Inofaddition,
addition,
the treated
Post-Hoc Post-Hoc
Post-Hoc LSD groups. LSD The
test test
test result of this study showed that 2 might 22 might
cell
showed death. In addition, Post-Hoc LSD test showed fibroblast
have ability
have atconcentration
ability cells.
ability
to induce This
to induce
induce
of phenomenon
fibroblast fibroblast
62.5fibroblast
µg/ml cell or maycell
death. have
higher death. clinical
Themight The
showedgroup
showed aa significant
treated
a significant significant
with
difference62.5difference
differenceµg/mlcalcium
(p<0.05) (p<0.05)
(p<0.05)between between
hydroxide
between have to cell death. The
acontrol
significantgroup difference
with all of(p<0.05)
the treatedbetween groups. control
The result implications
result
cause cell
revealed
result revealedas
revealed
death calcium
that in hydroxide
that
Vero
calcium
that calcium
fibroblast
calcium is widely
hydroxide cells. used
hydroxide
hydroxide Thisin
showed
control group group
control awithsignificant
all
with of all the difference
oftreated
the treated with groups.
groups. theThe groups The
group
group with
treated all of the
withµg/ml treated
62.5 µg/mlcalcium groups.
µg/mlcalcium The group
hydroxide dental
phenomenon
Noatconcentration treatments
atconcentration
atconcentration of may 62.5 of
4
.
of haveThe
62.5 reported
62.5 clinical
µg/ml µg/ml
or higher
µg/ml orclinical
or higher
implications evidence
might might
higher might
as
grouptreated
treated
group with
with 125
treated 62.5
with µg/mlcalcium
62.5 and 250 hydroxide µg/ml.
hydroxide
treated
showed with
a 62.5μg/mldifference
significant calcium hydroxide with the showed
groups cause provides
cause
cell
cause cell
death
cellfactualdeath
in
death information
Vero in
in Vero
fibroblast
Vero about
fibroblast
fibroblastcells. the cytotoxic
cells.
This
cells. This
This
showed showed a significant
a significant difference difference with the withgroups the groups
treated atreated
significant
with with with
125 difference
125 µg/ml
µg/ml with the
µg/ml
and andgroups
250 250 treated
µg/ml. No with
µg/ml. No phenomenon
effect
phenomenon
phenomenonof this
maymedicament may have
have
may have13,clinical
clinical clinical
butimplications implications
littleimplications
is known as about as
as
treated 125 and 250 µg/ml. No
125μg/ml and 250μg/ml. No significant difference the underlying molecular mechanisms. 82

82 82
82

106
 Paramitta et al.
Table 1. Means and standard deviations of the
percentage of fibroblasts cell viability after calcium
hydroxide treatment.
Groups Cell Viability (%) + SD
Negative Control 99.842 + 4.014
Ca(OH)2 62.5µg/ml 77.59 + 6.525
Ca(OH)2 125 µg/ml 56.206 + 6.595
Ca(OH)2 250 µg/ml 49.49 + 13.530
Fibroblast cell death mechanism induced by
calcium hydroxide in this study has not been clarified.
It was probably caused by the alkaline pH of calcium
hydroxide, which is almost 12.5-12.815. Based on
the previous in vitro study, pure calcium hydroxide
has high initiation pH14 and that the hydroxyl ions
are easily released in pure calcium hydroxide than
in other preparations16. The pH can influence some
of the cellular process such as cell metabolism,
the changes of the cell’s shape and mobility, the
adjustment of transportation process and cell
growth, cell proliferation and growth, conductivity
and transport through the cell membrane, and
isoosmotic cellular volume17.
The cell death mechanism caused by high alkaline
condition is not clearly known, but the previous study
demonstrated that mitochondria took an important
part in this cell death mechanism. The mitochondrial
damage was the initiation of cell death mechanism
caused by alkaline condition. This condition induced
the increase of mitochondrial trans-membran
potential leading to the disfunction of mitochondria.
Under normal condition, mitochondria is responsible
for cell respiration and therefore maintains the
proton gradient. The application of calcium
hydroxide may cause an alkaline condition, and it
may attract the proton away from mitochondria. The
mitochondria adapts by increasing respiration which
leads to the increase of radical superoxide. Under
these circumstances, the level of mitochondrial
trans-membrane potential elevation causes the
increase of superoxide diffusion from cytosol to the
mitochondrial membrane18. Another study suggests
that the increasing radical superoxide may cause
the de-energization of mitochondria and loss of
energy stores, peroxidation, and disruption of lipid
membranes, direct DNA damage, and activation of
transcriptional factor sactivation of protein-1 (AP-1)
and nuclear factor kappa beta (NF-κB) both of which
promote transcription of cytokines and have been
associated with induction of apoptotic cell death19.
There may be complicated molecular pathways
involved in calcium hydroxide-induced cell death.
It was reported that calcium hydroxide is able to
induce the increase of intracellular calcium ion in
Ca(OH)2-pretreated human pulp cells20. In fact the
increase in intracellular calcium level can generate
board spectrum of cellular processes21.
Conclusion
From our study it isconcluded that in vitro application
of calcium hydroxide at certain concentration may
cause fibroblast cell death, which may in part involve
in pulp tissue destruction.
Acknowledgements
We would like to thank Mrs. Istini for the help during
the experiment and also the staff of the Integrated
Research and Testing Laboratory (LPPT) Unit II,
Universitas Gadjah Mada, where this experiment
was conducted.
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