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.

University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 52

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 Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 56

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 : amit_kgdu@rediff.com