Keywords
benzalkonium chloride - chloride - pH - root canal sealers - solubility
Introduction
Infected root canals may contain many different microbial strains, mostly Gram-negative
anaerobes.[1]
[2]
[3] The most frequent species of bacteria detected in the root canal are Enterococcus faecalis, Streptococcus anginosus, Bacteroides gracilis, and Fusobacterium nucleatum.[4]
The root canal sealers come into direct contact with the remaining microorganisms
in the dentinal tubules and the inaccessible parts of the root canal system.[5]
[6] Therefore, the antimicrobial characteristics of these agents are desirable.[7]
One of the principal requirements of a sealer is that it should be bacteriostatic
or at least not encourage bacterial growth. The sealers play an important role in
sealing the root canal system by the entombing the remaining microorganisms and filling
the inaccessible areas within the prepared canals. Therefore, a sealer with antimicrobial
activity is highly beneficial in the elimination of the remaining microorganisms present
in the root canal after chemomechanical debridement and preventing the reinfection.
Some root canal sealers are known to be inherently antimicrobial, a feature which
can help to control the microorganism population.[8]
[9] However, these antimicrobial properties are generally short term and rarely extend
beyond 1 week,[9] which is insufficient to provide protection against persistent bacterial infection
in a clinical situation.[10]
Different antimicrobial agents (such as chlorhexidine, cetylpyridinium chloride [CPC],
and benzalkonium chloride [BC][11]
[12]
[13]) have been used in order to improve the antimicrobial characteristics of various
dental materials.
CPC is a quaternary ammonium compound which has a strong surface activity, and its
effect on the reduction of plaque and calculus has been demonstrated previously.[14] It has been widely used as an active component of oral antiseptics, and it is known
to have broad-spectrum antimicrobial properties, with a strong bactericidal effect
on Gram-positive pathogens and yeasts in particular.[13] The mechanism of action of CPC is that when it is in contact with the bacterial
cell membrane, inhibition of the cellular functions and cell death (bacteriolysis)
occurs.[14]
BC is recognized by the United States Pharmacopoeia as an auxiliary antimicrobial
agent recognizes.[14] It is the major antimicrobial component in numerous toothpastes and mouthrinses
as well as in dental restoratives,[15] and it is active against bacteria as well as certain viruses, fungi, and protozoa.[16]
The purpose of the study was to determine whether the addition of CPC and BC to root
canal sealers has an impact on the dissolution of tested materials, the release of
chloride ions (Cl—), and the pH value.
Materials and Methods
Three commercial materials for root canal sealing were used ([Table 1]). Three sets of six samples were prepared from each material: (a) without addition
of an antimicrobial agent, (b) addition of 2% by weight of CPC, and (c) addition of
2% by weight of BC.
Table 1
Materials used in the study
|
Materials
|
Manufacturer
|
|
EndoRez
|
Ultradent Products, Inc. (South Jordan, UT, United States)
|
|
N2
|
Hager and Werken GmbH and Co. KG
|
|
Apexit Plus
|
Ivoclar Vivadent AG, Schaan, Liechtenstein
|
The samples were prepared by mixing the components according to the manufacturers’
instructions, following the method as described by Dimkov et al.[16] The samples with the antimicrobial agents were prepared by mixing 2% weight of the
antimicrobial substances to the newly mixed cement. The resulting paste was then placed
in metal molds with dimensions 6 mm (height) and 4 mm (diameter). Sample-filled molds
were placed in an incubator with constant temperature of 37°C for a period of 24 h.
Then, each sample was stored in 5-mL distilled water. All samples were tested at 3
time intervals: 1 day, 1 week, and 1 month following their storage in distilled water.
Examination of the pH Value of the Storage Solution
The pH value is a measure of activity of hydrogen ions (H+) in the solution that determines whether a given solution is acid or base. The pH
value is measured based on the activity of hydrogen ions within the solution.[8] The formula to calculate pH is as follows:
The pH meter is an electrical instrument used to measure the pH value of liquids.
A typical pH meter is made of a special glass electrode, connected with an electronic
measuring device. The test was performed with the use of a digital pH meter (Whatman
PHA2000).
At the very beginning, the instrument was calibrated with standard solutions with
predetermined pH value (pH = 7.0 and pH = 4.0), and subsequently, the pH of the storage
solutions was measured after the storage time intervals.
Examination of the Solubility of the Material
The solubility of the material was determined by analytical laboratory balance (Sartorius,
A&D GR-202 Semi-Micro-Balance) with an accuracy of 0.0001 g. The test was conducted
by measuring the mass of samples before and after expiration of the predetermined
time intervals.
Release of Cl—
The examination of the amount of free Cl— in the tested solutions was performed by an ion-selective electrode, specific for
the chloride (ORION 4-Star pH-ISE Benchtop instrument, Thermo Electron Corporation).
The electrode is designed to respond to chloride ions in aqueous solutions.[7]
The test starts by an instrument calibration with NaCl solutions with predetermined
concentrations (0.1 ppm, 1.0 ppm, 10.0 ppm, 100.0 ppm, and 1000.0 ppm NaCl); then,
a calibration curve is prepared and followed by measurement of the released Cl— of the storage solution.
Statistical Analysis
The statistical analysis was performed by one-way analysis of variance and the post
hoc Tukey's honestly significant difference test, in STATISTICA 7.1 (TIBCO Software
Inc., Palo Alto, CA) and SPSS 17.0 (SPSS, IBM, NY, United States) platform for Windows.
The level of significance was set at P < 0.05.
Results
The release of Cl— from the materials is an indicator for the increase of their antimicrobial activity.
The results obtained in regard to the release of Cl
—
([Table 2]) from Apexit, N2, and EndoRez (without the addition of antimicrobials) indicate
the presence of Cl— in the solutions following the predetermined storage intervals. Apexit Plus shows
highest release level after day 1, although all the tested materials with the addition
of an antimicrobial agent show higher level of released Cl— compared to the Apexit samples without incorporation of antimicrobials. The previous
findings are an indicator that the addition of antimicrobial agents leads to an increased
release of Cl—.
Table 2
Average values and standard deviations of measures for the release of Cl— ions in the materials: Apexit Plus, N2, and EndoRez (without and with addition of
benzalkonium chloride and cetylpyridinium chloride)
|
Chloride ions (ppm)
|
Materials used
|
Mean (SD)
|
p-Value
|
|
1 day
|
1 week
|
1 month
|
|
Abbreviations: BC, benzalkonium chloride; CPC, cetylpyridinium chloride; SD, standard
deviation. The difference between the arithmetic means is statistically significant
at P > 0.05.
a1 day–1 week.
b1 day–1 month.
c1 week–1 month.
|
|
Cl— ions (ppm)
|
Apexit
|
0.07 (0.03)a,b
|
0.02 (0.00)a
|
0.02 (0.00)b
|
0.00
|
|
Apexit + BC
|
0.14 (0.01)a,b
|
0.08 (0.02)a,c
|
0.03 (0.010)b,c
|
0.00
|
|
Apexit + CPC
|
0.96 (0.02)a,b
|
0.01 (0.00)a,c
|
0.06 (0.03)b,c
|
0.00
|
|
Cl— ions (ppm)
|
N2
|
0.25 (0.03)a,b
|
0.15 (0.02)a
|
0.12 (0.02)c
|
0.00
|
|
N2 + BC
|
0.34 (0.01)b
|
0.32 (0.04)
|
0.27 (0.02)b
|
0.01
|
|
N2 + CPC
|
0.33 (0.04)b
|
0.29 (0.01)
|
0.26 (0.03)b
|
0.00
|
|
Cl— ions (ppm)
|
EndoRez
|
0.07 (0.01)a,b
|
0.02 (0.02)a
|
0.02 (0.01)b
|
0.00
|
|
EndoRez + BC
|
0.15 (0.03)a,b
|
0.02 (0.00)a
|
0.02 (0.00)b
|
0.00
|
|
EndoRez + CPC
|
0.43 (0.09)a,b
|
0.02 (0.00)a
|
0.02 (0.00)b
|
0.00
|
The endodontic cements without BC and CPC show an increase in mass after 1-month storage
([Table 3]), except EndoRez which reduces its mass. When BC was added, only EndoRez marked
an increase in the mass. When CPC was added, Apexit Plus and N2 decreased in weight
after 1 month compared to EndoRez that did not alter its mass.
Table 3
Average values and standard deviations of the mass of Apexit, N2, and EndoRez (without
and with addition of benzalkonium chloride and cetylpyridinium chloride)
|
Mass (g)
|
Materials used
|
Mean (SD)
|
p-Value
|
|
At the beginning
|
After 1 month
|
|
The difference between the arithmetic means is statistically significant at the level
P > 0.05. a1 day–1 week, b1 day–1 month, c1 week–1 month. BC, benzalkonium chloride, CPC, cetylpyridinium chloride, SD, standard
deviation.
|
|
Mass (g)
|
Apexit
|
0.09 (0.01)
|
0.10 (0.01)
|
0.04
|
|
Apexit + BC
|
0.09 (0.02)
|
0.09 (0.01)
|
0.82
|
|
Apexit + CPC
|
0.11 (0.01)
|
0.10 (0.01)
|
0.07
|
|
Mass (g)
|
N2
|
0.12 (0.01)
|
0.14 (0.01)
|
0.01
|
|
N2 + BC
|
0.15 (0.01)
|
0.15 (0.03)
|
0.99
|
|
N2 + CPC
|
0.15 (0.02)
|
0.14 (0.02)
|
0.29
|
|
Mass (g)
|
EndoRez
|
0.11 (0.01)
|
0.10 (0.02)
|
0.42
|
|
EndoRez + BC
|
0.10 (0.03)
|
0.11 (0.03)
|
0.64
|
|
EndoRez + CPC
|
0.10 (0.02)
|
0.10 (0.02)
|
0.87
|
The pH value of Apexit Plus gradually increased between the first day and the end
of the first month. In the sealers with incorporation of BC and CPC, the pH gradually
increased in the first week, whereas after 1 month, the pH value lowered ([Table 4]). The solutions where N2 samples were stored did not significantly change their
pH value. The solutions where the EndoRez samples without addition of antimicrobials
were stored showed a significant continuous reduction (P < 0.01) of the pH values, while the solutions from EndoRez samples with addition
of antimicrobials these changes were statistically significant at the level of P < 0.01.
Table 4
Average values and standard deviations of the pH value of the solutions with Apexit,
N2, and EndoRez (without and with addition of benzalkonium chloride and cetylpyridinium
chloride)
|
pH value
|
Materials used
|
Mean (SD)
|
P-Value
|
|
1 day
|
1 week
|
1 month
|
|
Abbreviations: BC, benzalkonium chloride; CPC, cetylpyridinium chloride; SD, standard
deviation. The difference between the arithmetic means is statistically significant
at the level P > 0.05.
a1 day–1 week.
b1 day–1 month.
c1 week to–1 month.
|
|
pH
|
Apexit
|
7.46 (0.11)
|
8.41 (0.87)
|
8.65 (0.87)
|
0.35
|
|
Apexit + BC
|
7.69 (0.03)a,b
|
9.35 (0.00)a,c
|
8.13 (0.00)b,c
|
0.00
|
|
Apexit + CPC
|
7.48 (0.14)a,b
|
9.02 (0.08)a,c
|
8.03 (0.08)b,c
|
0.01
|
|
pH
|
N2
|
6.58 (0.36)
|
6.95 (0.25)
|
6.93 (0.25)
|
0.46
|
|
N2 + BC
|
6.56 (0.09)
|
6.74 (0.15)
|
6.58 (0.15)
|
0.44
|
|
N2 + CPC
|
6.39 (0.04)
|
6.54 (0.28)
|
6.52 (0.28)
|
0.79
|
|
pH
|
EndoRez
|
6.78 (0.01)a,b
|
6.65 (0.01)a,c
|
6.55 (0.01)b,c
|
0.00
|
|
EndoRez + BC
|
6.5 (0.04)a,b
|
6.35 (0.01)a,c
|
6.87 (0.01)b,c
|
0.01
|
|
EndoRez + CPC
|
5.83 (0.06)a,b
|
4.75 (0.07)a,c
|
6.83 (0.01)b,c
|
0.00
|
Discussion
A study by Dimkov et al was performed by adding BC and CPC in two glass-ionomer cements,
and the results showed that the release of Cl— is linear to the increase of the concentration of the solutions.[16] These results are in line with the current study, where the tested endodontic cements
release Cl—, although with lower values. Once antimicrobial agents have been added, the values
of released Cl— increased.
Another study suggests that the sealers based on calcium hydroxide show high solubility.[17] Mushtag et al studied the solubility of some endodontic materials during removal
from the canal system using several types of solvents: xylene, refined orange oil,
and tetrachloroethylene; distilled water was used as a control. Apexit Plus is most
soluble in xylene and then in refined orange oil and tetrachloroethylene.[18] The results obtained in this study are in accordance with the previous studies and
prove that the highest level of solubility was found in Apexit Plus.
According to this research, the largest stability related to the solubility was demonstrated
by EndoRez, which is basically a positive feature, because it does not lead to progressive
loss of material.
The root canal sealers based on zinc oxide are frequently used in endodontics (with
or without additions). In this study, N2 has been used. Along with the zinc oxide,
it contains traces of paraformaldehyde in order to improve its antimicrobial activity.
Previous studies indicate that this agent has its own inherent antimicrobial activity
toward the different types of micro-organisms even without the addition of an antimicrobial
agent.[19]
[20] However, the current examination demonstrated that N2 following addition of BC and
CPC shows a statistically significant increase in the release of Cl— (and consequent improvement of the antimicrobial effect) on a statistically significant
level of P < 0.01.
In the study by Flores et al, antibacterial activity of four different root canal
sealers against bacteria commonly found in endodontic infections were tested. The
results suggested that the materials based on zinc oxide have highest antibacterial
activity, while EndoRez showed lowest antibacterial activity of all tested materials.[21]
The current results obtained for the release of chloride ions from Apexit, N2, and
EndoRez (without the addition of antimicrobial agents) indicate the presence of Cl— in solutions following different storage intervals. The addition of antimicrobial
agents increases the release of Cl—, which is considered as an indicator of a stronger antimicrobial activity.
The increase of the pH value of the solutions in which endodontic cements are stored
is directly linked to their antimicrobial activity, which is supported by the results
of several previous studies, where the increase of the pH value of the solutions results
with an increase in their antimicrobial activity.[22]
[23] Solutions where N2 samples were stored did not have a significant change in their
pH value. The EndoRez samples demonstrated a significant continuous reduction of the
pH values; therefore, in EndoRez solutions, these changes are important because they
will lead to a decrease in the antimicrobial activity. Consequently, the addition
of BC and CPC, especially in the first week, is beneficial because it leads to lower
pH values and an increase in the antimicrobial activity.
Conclusions
The addition of antimicrobials leads to improved characteristics of the root canal
sealers. After addition of antimicrobials, the release of Cl— increases and the level of pH decreases, which are indicators of stronger antimicrobial
activity.
