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ABSTRACT : Aims And Objectives: To compare calcium ion, hydroxyl ion release and pH
levels between nano calcium hydroxide and other calcium hydroxide based intracanal
medicaments in vitro.
Materials And Methods: Calcium hydroxide based substances were divided into six groups
(n=5): Group A (Calcium hydroxide powder with distilled water), Group B (Nano calcium
hydroxide powder with distilled water), Group C (Vitapex), Group D (RC Cal), Group E
(Dentocal) & Group F (Calcium hydroxide points). For determining the particle morphology of
different calcium hydroxide products, TEM technique was used. 30 polyethylene cylindrical
tubes were taken, one end of which was sealed with temporary material and filled with the
materials of each group. Tubes were then immersed in separate calibrated beakers, each with 10
ml of distilled water. For each group, 5 samples were analyzed after 0, 10, and 20 minutes; and
after 1, 2, 24 and 48 hours, 1 week and 1 month. The pH value was measured by calibrated pH
meter. Ion liberation was measured by an ICP-atomic emission spectrometry. Statistical analysis
was done by ANOVA and Tukey's post hoc tests.
Results: Group B had the lowest particle size. According to calcium ion and hydroxyl ion
liberation, RC Cal was better and according to pH value, Dentocal was better than other groups.
Conclusion: Aqueous based preparations of calcium hydroxide should be chosen over points or
oil-based calcium hydroxide preparations.
1 2 3 4 5Sonali, Garg Amit Kumar, Paul Rohit, Hans Manoj, Nagpal Ajay
Department of Conservative Dentistry and Endodontics
K. D. Dental College and Hospital, Mathura
INTRODUCTION: Calcium hydroxide, widely used in
endodontics is a strong alkaline substance having pH nearly
12.5. It dissociates into calcium and hydroxyl ions in aqueous
medium. Antimicrobial activity, inhibition of tooth resorption
and induction of repair by hard tissue formation are its
biological properties. (1-3) That's why it has been
recommended for use in several clinical situations. (4)
Release of hydroxyl ions as highly oxidant free radicals show
extreme reactivity with several biomolecules. (5)
The microorganisms penetrate into infected dentin tubules
from 50 to 100 ìm depth. (6) The application of calcium
hydroxide into instrumented and irrigated root canals
eliminates microorganisms effectively is known. (7) Due to
buffering capacity of hydroxyapatite selective permeability
of the hydroxide ions in the dentin tubules is there. (8)
Because of a high local pH particles inserted into the open
dentin tubules may act as a direct source of dissociated
calcium hydroxide which dissolve continuously in aqueous
form, enhancing antimicrobial effectiveness. (9)
Nano-particles are microscopic particles of less than 100 nm
which are different in properties such as active surface area,
chemical and biological reac- tivity. (10) They are getting
popular in dentistry and medicine as antibacterial agents. The
higher surface to volume ratio and charge density results in
their greater interaction with the environment and thus causes
a higher antibacterial activity. (11) So this study was done to
find out the effect of nano-particles & other calcium
hydroxide based intracanal medicaments on the release of
calcium & hydroxyl ions and on pH levels.
COMPARATIVE EVALUATION OF CALCIUM ION, HYDROXYL ION RELEASE AND PH LEVELS BETWEEN NANO CALCIUM HYDROXIDE AND OTHER CALCIUM HYDROXIDE BASED INTRACANAL MEDICAMENTS : AN IN VITRO STUDY
Keywords :
Calcium hydroxide,
pH, Calcium ions,
Hydroxyl ions
Source of support : Nil
Conflict of interest: None
Journal of Dental Sciences
University
University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 50
University J Dent Scie 2017; No. 3, Vol. 1
ResearchArticle
MATERIALS AND METHOD : This study was carried out
in the Department of Conservative Dentistry and
Endodontics, K. D. Dental College and Hospital, Mathura,
Uttar Pradesh, in the Department of University Sophisticated
Instrument Facility, Aligarh Muslim University, Aligarh,
Uttar Pradesh and at the Atmy Analytical Labs Private
Limited, DLF Industrial Area, Faridabad, Haryana.
Calcium hydroxide based substances were divided into six
groups (n=5):
·Group A: Calcium hydroxide powder with distilled water
(Sigma-Aldrich, St.Louis, Missouri, United States)
Group B: Nano calcium hydroxide powder with distilled
water (Pankaj Enterprises, New Delhi, India)
Group C: Vitapex (J. Morita, Tokyo, Japan)
Group D: RC Cal (Prime Dental Products, Mumbai, India)
Group E: Dentocal (Anabond Stedman Pharma Research,
Chennai, India)
Group F: Calcium hydroxide points (Coltene whaledent,
Mahwah, NJ, USA)
For determining the particle size and shape of different
calcium hydroxide products TEM (transmission electron
microscopy) (JEOL, JEM-2100Plus, USA) technique was
used. Measurements were performed on the samples dried
under vacuum.
Thirty polyethylene cylindrical tubes were taken, one end of
which was sealed with 1 mm layer of temporary material
(Orafil-GTM, Prevest DenPro, Jammu, India) and filled with
the respective materials of each group (n=5).
An endodontic file was used to introduce carefully calcium
hydroxide pastes into the tubes through the opening, avoiding
bubble formation. RC Cal, Dentocal and Vitapex were placed
according to manufacturer's instructions. Calcium hydroxide
points (#40) were introduced until tubes were completely
filled.
Tubes were then immersed immediately in separate calibrated
beakers, each with 10 ml of distilled water with neutral pH
which was used as extraction solution. The extraction solution
was maintained at room temperature and without agitation.
For each group, 5 samples were analyzed. For each group,
samples were analyzed after 0, 10, and 20 minutes; and after 1,
2, 24 and 48 hours, 1 week and 1 month.
The pH value was measured with a calibrated pH meter
(Eutech Instruments, Ayer Rajah Crescent, Singapore).
Calcium ion liberation was measured by an inductively
coupled plasma-atomic emission spectrometry (ICP-AES) at
same time intervals used for pH readings. ICP spectrometer
(Perkin Elmer USA Model Optima 3300RL) was used at a
wavelength of 317.93 nm specific for calcium quantification.
From the calculation of calcium ions liberated, it was possible
to determine the amount of hydroxyl ions liberated. The
molecular weight of 2 mol hydroxyl ion is 34, and molecular
weight of 1 mol calcium ion is 40.08, and molecular weight of
complete molecule is 74.08. The percent of the two in the total
weight was calculated to be 45.89% and 54.11%,
respectively. Therefore in 1 mol of calcium hydroxide, there
was 45.89% hydroxyl ions and 54.11% calcium ions, the
quantity of hydroxyl ions was calculated as they were directly
proportional.
The antimicrobial action of calcium hydroxide depends on the
concentration of hydroxyl ions in the solution. Then it was
also analyzed that whether particle morphology do have any
effect on the pH, calcium ion release and hydroxyl ion release.
Statistical analysis was carried out using one way analysis of
variance (ANOVA) and Tukey's honestly significant
difference (HSD) post hoc tests with PASW statistics version
18 to compare the statistical difference. Significance level
was set at P-value less than 0.05.
RESULTS:
Figures: TEM images of A) Calcium hydroxide (Sigma-
Aldrich) powder showing irregular particle morphology. B)
Calcium hydroxide (nano) powder showing circular particle
morphology. C) Vitapex showing circular particle
morphology. D) RC Cal showing circular particle
morphology. E) Dentocal showing irregular particle
University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 51
University J Dent Scie 2017; No. 3, Vol. 1
morphology. F) Calcium hydroxide points showing irregular
particle morphology.
Mean particle size (nm) of different calcium hydroxide
intracanal medicaments in TEM represented the largest
particle size of Group C followed by Group D, E, A and F
respectively. While, Group B having the lowest particle size
as revealed by One-Way ANOVA.
Table 1. Mean pH of Different Calcium Hydroxide Intracanal
Medicaments.
F=ANOVA Test
N=Sample Size
Table 1 represents that at 0 min, 10 min & 20 min, Group C
reached the higher pH value than all other groups. At 1 hr, 2
hrs, 24 hrs, 1 week & 1 month, Group E reached the highest
pH than other groups. At 48 hrs, Group D reached the highest
pH than other groups. At 0 min & 10 min, Group A had least
pH value than all other groups. Group B had least pH value at
20 min, 1 hr, 2 hrs & 1 week.
Table 2. ICP–AES Mean Calcium Ion Release (ppm) of
Different Calcium Hydroxide Intracanal Medicaments.
F=ANOVA test
N=Sample Size
Table 2 represents that calcium release was highest in Group
D followed by Group E at all time intervals. Group A had least
calcium release at 0, 20 min, 1 hr, 2 hrs, 1 week & 1 month but
higher than Group F at 10 min, 24 hrs & 48 hrs.
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University J Dent Scie 2017; No. 3, Vol. 1
Table 3. Mean Hydroxyl Ion Release (ppm) of Different
Calcium Hydroxide Intracanal Medicaments.
F=ANOVA Test
N=Sample Size
Table 3 represents that hydroxyl ion release was highest in
Group D followed by Group E at all time intervals. Group A
had least hydroxyl ion release at 0, 20 min, 1 hr, 2 hrs, 1 week
& 1 month but higher than Group F at 10 min, 24 hrs & 48 hrs.
DISCUSSION : Calcium hydroxide aqueous paste, widely
used as interim antimicrobial dressing in root canal treatment
dissociates at body temperature into calcium ions (Ca2+) and
hydroxide ions (OH- ), leaving mostly particles undissolved.
(12) The antibacterial action is by the control of bacterial
enzymatic activity. (13) The release of hydroxyl ions raises
the pH of the medium inactivating the essential enzyme
system of bacteria. The mineralizing effect can be explained
by the activation of alkaline phosphatase enzyme due to its
elevated pH. (13) The best pH for the activation of this
enzyme ranges from 8.6 to 10.3. (14)
The permeability of dentin is due to tubule anatomy, density,
diameter, and length as well as size and charge, studied by
various investigators. (15, 16) Mjor et al. reported that the
tubules were irregular in direction and density at apical root
dentin having a diameter of 2 to 5 ìm. (15) Dentin is a
substrate, whereas calcium hydroxide is a material and the
size of the dentin tubules correlates with the size of the
calcium hydroxide particles. The geometry of the small
particles allow calcium hydroxide to enter the open dentinal
tubules. The TEM images in our study revealed that
morphology of different particles of Group A, E & F was
irregular whereas that of Group B, C & D was circular. Also it
was found that width of the particles of nano calcium
hydroxide was less than 10 nm and had the least value among
all the groups. These results were in agreement with the study
performed by Yasaei M et al. (17)
Taglieri G et al. (18) studied calcium hydroxide nanoparticles
in aqueous suspensions structurally and morphologically with
X-Ray diffraction (XRD) and transmission electron
microscopy (TEM), respectively and found that they were
crystalline, regularly shaped, hexagonally plated with
dimensions 30 nm to 300 nm or less. In an another study under
TEM, (19) he found the presence of linear chains constituting
very small particles of diameters less than 10 nm. Presence of
very small particles was because of the higher amounts of
surfactant. It was found that undissolved fine particles may
cause antimicrobial action inside dentin tubules.
For effective therapeutic action of calcium hydroxide
preparations, time seems to be a vital factor. Zmener et al.
tested the pH changes over a period of 30 days using a mixture
of calcium hydroxide and distilled water and two commercial
calcium hydroxide products in a simulated periapical
environment and found that there was a rapid increase in the
pH at l hour and 24 hours, followed by continuous but more
gradual increase from 15 to 30 days. (20) In our study all the
University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 53
University J Dent Scie 2017; No. 3, Vol. 1
groups reached at maximum pH at one week which gradually
decreased with time as observed after one month.
Many substances have been added to the calcium hydroxide
powder to improve its properties such as the antibacterial
action, radiopacity, flow, and consistency. The ideal vehicle
should allow a gradual and slow release of calcium and
hydroxide ions. The vehicles suggested can be classified as
aqueous and oily. Safavi et al. studied the effect of mixing
vehicle on dissociation of calcium hydroxide in solution.
They found that the use of non-aqueous mixing vehicles may
impede the effectiveness of calcium hydroxide as a root canal
dressing. (21) Similarly to our study Vitapex having oily
vehicle presented less effectiveness in both pH and release of
calcium and hydroxyl ions than aqueous vehicles.
Carvalho CN et al. (22) evaluated pH and release of calcium,
sodium and phosphate ions from different medications in
human dentin and measured ions by (ICP/AES) at 10 min, 24
h, 7, 14, 21 and 30 days. It was found that CH had the highest
level of pH & calcium ions release at 30 days because of
different vehicles used and the concentration of chlorhexidine
which may affect its ability to release ions. (23) Similarly in
our study also mixing of vehicles had an effect on release of
ions and pH. But in our study release of ions rapidly increased
after 2 hrs, reached maximum value after 1 week and
gradually decreased till 1 month.
Pawinska M et al. (24) evaluated in vitro release of hydroxyl
ions from several calcium hydroxide preparations –
nonsetting and setting canal sealers and points. It was found
that nonsetting preparations have a significantly higher
capability of hydroxyl ions release. The pH values of samples
of all materials correlated positively with time. Almost all
materials reached a maximum on the 8th day of the
experiment same as in our study.
Eppendorf tubes were preferred over natural teeth because the
difference in size of apical foramina and anatomic variations
like cul-de-sacs fins and lateral canals etc. could have lead to
various dentinal tubules opening onto the root surface and
thus variable results could have achieved. (25)
RC Cal and Dentocal were used in the study along with
calcium hydroxide distilled water as they are highly alkaline,
non toxic and water-based radiopaque calcium hydroxides
pastes with barium sulphate in ready to use paste form which
could be easily cleaned and removed from canal whenever
required. They have better chemical stability and non drying
nature. They are both antibacterial and bacteriostatic in
nature. Barium sulphate in these pastes is added in order to
increase the radioopacity.
Estrela et al. described liberation of calcium and hydroxide
ions faster and more significant when calcium hydroxide
distilled water paste was used. (26) The results of present
study favour more liberation of calcium and hydroxyl ion
from the RC Cal and Dentocal pastes as compared to calcium
hydroxide with distilled water paste. This difference may be
caused by the different methodologies used as well as
addition of barium sulphate in these pastes. According to
Sahrawat KL et al., the ICP-AES method provided a better
precision than the colorimetric method for the determination
of hot water extractable B in the soil samples. (27) ICP-AES
method describes multi-elemental determinations by using
sequential or simultaneous optical systems and axial or radial
viewing of the plasma. The instrument measures
characteristic emission spectra by optical spectrometry.
Samples are nebulized and the resulting aerosol is transported
to the plasma torch. Element-specific emission spectra are
produced by a radio-frequency inductively coupled plasma.
The spectra are dispersed by a grating spectrometer, and the
intensities of the emission lines are monitored by photo-
sensitive devices. (28) So we used the ICP-AES method in the
present study.
A gutta-percha point is newly introduced for calcium
hydroxide delivery with fewer residues, composing 52%
Ca(OH)2, 42% gutta percha, sodium chloride, surfactant and
coloring agents. They are the hygienic time release
preparations releasing Ca(OH)2 from a gutta percha matrix.
They are firm yet flexible for easy introduction into root
canal. Pure calcium hydroxide is homogenously distributed
throughout a gutta percha matrix. Sodium chloride and
surfactant improve the solubility of calcium hydroxide and
mobility of ions. They are ISO standard and have light brown
color to avoid confusion with gutta percha points. A drop of
sterile water may be used together with the point for initial
release of ions. However after insertion into root canal,
sufficient fluid flows into space between point and canal wall
from dentinal tubules and apical area to activate the Ca(OH)2
even without additional water. Due to low solubility of
calcium hydroxide only small amounts are released at one
time. The surrounding fluid is quickly saturated. However, as
further moisture flows into the canal calcium hydroxide is
continuously released maintaining high pH.
Economides et al. evaluated the release of hydroxyl ions and
found that calcium hydroxide containing gutta-percha points
showed a significantly lower alkalinizing potential than the
University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 54
University J Dent Scie 2017; No. 3, Vol. 1
non-setting preparation and calcium hydroxide mixed with
distilled water. (29) The gutta-percha matrix probably binds
the hydroxyl ions and blocks their release at the site of
application. In our study also the pH was not significantly
increased by gutta-percha point. Economides et al. and
Azabal-Arroyo et al. reported maximum pH values of 9.5 and
10.9, respectively. (30) Calt et al. showed that calcium
hydroxide gutta-percha points did not induce any changes in
pH and calcium ion levels. (31) Larsen and Horsted - Bindslev
concluded that hydroxyl ion liberation from the gutta-percha
points is limited compared to that from calcium hydroxide
pastes. These results are comparable to those of our study.
Lohbauer et al. evaluated calcium ion release and pH
characteristics of calcium hydroxide plus points (new
formula, greater dissociation and even more effective) and
found that calcium hydroxide plus points had a greater release
of calcium ions compared to conventional calcium hydroxide
points. (32) This result is due to greater dissociative ability of
calcium hydroxide plus points having similar composition as
compared to conventional calcium hydroxide points. The
diffusion of hydroxyl ions through dentin from different
calcium hydroxide medicaments was determined by Sevimay
et al. (33) They found that non-setting calcium hydroxide
based materials have an effective release of hydroxyl ions
compared with calcium hydroxide plus points. The results of
present study are comparable to those observed in previous
studies.
Vitapex (Yellow soft paste with iodoform odor) contains
calcium hydroxide with iodoform in silicon oil with excellent
accessibility. It is composed of 40.4% iodoform, 22.4%
silicone oil, 6.9 % inert substances and calcium hydroxide.
Iodoform provides bacteriostatic property and increased
radioopacity. Silicone oil act as lubricant and ensures
complete coating of canal walls. Due to inert substances it
never hardens and gets solubilized as well as calcium
hydroxide remains active in root canal. Also it remains
chemically stable. It was observed that oil paste containing
calcium hydroxide was largely lacking in both ion release and
antimicrobial properties. Larsen and Bindslev observed that
the aqueous suspension exhibited the highest pH and calcium
ion liberation. (34) The low solubility and poor ability to
diffuse make it difficult for oil paste containing calcium
hydroxide compounds to reach maximum pH levels in a short
period of time. The results of our in vitro study show that they
should be used for a minimum of 7 days to achieve maximum
therapeutic effectiveness.
On the other hand, nano calcium hydroxide showed least pH
which was in contrast with the fact that smaller particle size
leads to better penetration into dentinal tubules and rise in pH.
The present study showed that particle size did not have any
effect on the pH, Ca++ ion release & OH- ion release. Also
further investigations should be performed.
Hence, from this study it can be formulated that Non-setting
premixed calcium hydroxide pastes are better in antimicrobial
activity as compared to calcium hydroxide powder and
calcium hydroxide points.
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University J Dent Scie 2017; No. 3, Vol. 1
CORRESPONDING AUTHOR:
Dr. Amit Kumar Garg
Department of Conservative Dentistry and Endodontics
K.D. Dental College and Hospital, Mathura
Email : [email protected]