Incorporation of antimicrobial agents can be used to enhance the antibacterial effect of endodontic sealers

Abstract

Aim

The antibacterial activity of five endodontic sealers against three different microorganism strains alone and following incorporation of 2% benzalkonium chloride (BC) and 2% cetylpyridinium chloride (CPC) was evaluated.

Methodology

The agar diffusion method was used to determine the inhibitory effect of the following endodontic sealers: RoekoSeal, Endomethasone N, N2, Apexit Plus and AH plus, on Streptococcus mutans – ATCC 25175, Lactobacillus casei – ATCC 4646 and Actinomyces viscosus – ATCC 19246. Bacterial strains were inoculated into BHIB, and incubated in an anaerobic atmosphere (37 °C). From the bacteria grown in the liquid medium, the density of the inoculum was set to be equivalent to McFarland 2 standard. In Shaedler agar, 350 μL of the bacterial suspension were equally spread. Specimens (4 mm × 6 mm) were prepared from each material without and with addition of 2% BC or 2% CPC. The inhibition zones were determined after 2 days, after 7 days and after 21 days of incubation.

Results

The largest inhibition zones were shown at zero time in all cases, with progressively less inhibition at 7 and 21 days. Endomethasone N and N2 showed the most intense antimicrobial activity, while RoekoSeal showed the least antimicrobial effect. The most susceptible microorganism was A. viscosus . Greater antimicrobial effects were found following incorporation of BC or CPC, and generally, BC gave greater inhibition zones than CPC.

Conclusions

Adding either BC or CPC has the potential to improve clinical outcomes with endodontic sealers, as these substances enhance the short-term antimicrobial effects of the sealers.

Introduction

Infection of the root canal by microorganisms after endodontic treatment is undesirable, and reducing the population of infecting organisms is a key feature of successful treatment . This can be achieved by instrumentation, irrigation and intra-canal medication . Endodontic sealers are also known to have inherent antibacterial activity, which may help to control the population of micro-organisms .

The mouth is host to a variety of potentially damaging bacteria. For example, Streptococcus mutans is the primary species associated with dental caries , while deep caries and advanced root lesions are caused by the group of Gram-positive cocci and rods, predominantly streptococci, lactobacilli, Actinomyces , and yeasts . Infected root canals have been found to contain various bacteria, the majority of which are Gram-negative anaerobes .

The ultimate goal of root canal therapy might be considered to be complete elimination of all microorganisms from the root canal system. Unfortunately, this appears to be impossible in typical clinical situations . However, the use of endodontic sealers with antibacterial properties may help in reducing the number of infecting microorganisms and in eradicating infection . To this end there have been several studies aimed at assessing the antimicrobial activity of different endodontic sealers .

Antimicrobial properties are desirable in endodontic sealers as the sealer is likely to come in direct contact with any microorganisms remaining in the dentinal tubules and un-debrided parts of the root canal system . Control of the infection depends not only on removal of the bacteria from the root canal system but also on avoiding the re-infection through good canal obturation .

In principle it is possible to improve the antimicrobial properties of materials by the addition of antimicrobial agents. Within dentistry, several substances have been used, for example in mouthwashes, dental restorative materials and toothpastes. These include chlorhexidine , cetylpyridinum chloride and benzalkonium chloride .

Cetylpyridinium chloride (C 21 H 38 NCl) (CPC) is widely used as the active component of oral antiseptics and it is known to have a broad antimicrobial spectrum with a strong bactericidal effect on Gram positive pathogens .

The official US pharmacopoeia recognizes benzalkonium chloride (alkyldimethylbenzylammonium chloride) (BC) as an auxiliary antimicrobial agent . It is the major antimicrobial agent in numerous toothpastes and mouthrinses, as well as in dental restoratives and it is active against bacteria as well as certain viruses, fungi and protozoa .

The aim of the present study is to explore the effect of adding two of these substances, BC and CPC, to a selection of current endodontic sealers, and determining the effect against relevant microorganisms ( S. mutans , Lactobacillus casei , Actinomyces viscosus ). The null hypothesis tested was that the incorporation of BC and CPC did not affect the antibacterial properties of the tested endodontic sealers. The antibacterial behavior of the materials themselves has also been determined and compared with the antibacterial properties following incorporation of the additives (at 2% by mass in each case).

Materials and methods

Five commercial endodontic sealers were used in the study, namely RoekoSeal, Endomethasone N, N2, Apexit Plus and AH Plus. Three sets of 6 samples were prepared from each material as follows: (a) without antimicrobial agent, (b) with 2% (w/w) CPC and (c) with 2% (w/w) BC. All of the materials were tested at three time intervals, namely after 2 days, after 7 days and after 21 days of incubation.

Specimen preparation

Samples were prepared by mixing the components according to the manufacturers’ instructions. The samples with antimicrobial agents were prepared by mixing 2% by mass of the antimicrobial (CPC or BC, respectively) into the newly mixed cement. The paste was then placed in metal molds of dimensions: 6 mm (height) by 4 mm (diameter). The molds were closed by metal plates on both sides and clamped. The molds were placed in an incubator at 37 °C for 24 h and the specimens allowed to set.

Microbiological analysis

The agar diffusion method was used for the determination of the inhibitory effect of the specimens on 3 different test strains: S. mutans – ATCC 25175, L. casei – ATCC 4646 and A. viscosus – ATCC 19246 (manufactured by American Type Culture Collection, Manassas, VA, USA).

The bacterial strains were inoculated into BHIB (Brain Heart Infusion Broth, Oxoid, Wesel, Germany) and incubated in an anaerobic atmosphere at 37 °C for 48 h. From the bacteria grown in the liquid medium, the density of the inoculum was set to be equivalent to the McFarland 2 standard. Then, in previously marked Shaedler agar with addition of sheep blood (Schaedler + SB, Oxoid, Wesel, Germany), 350 μL of the bacterial suspension were inoculated.

After the inoculation, and a subsequent period of 15 min for absorption of the bacterial suspension by the agar plate; sterile plastic tubes were used to create wells (3 mm depth and 3.5 mm width) into the agar plate. A total of 7 wells were prepared on each dish, 6 along the perimeter and one in the center. The distance between the wells was 30 mm, and the distance between the wells and the dish wall was 15 mm. Then, the specimens were carefully placed in the wells with sterile tweezers, so that they would have close contact with the agar. The Petri dishes were left for 30 min at room temperature, and incubated anaerobically at 37 °C for 48 h. The anaerobic atmosphere for cultivation was produced by anaerobic pots, with an atmosphere of 10% of carbon dioxide and 90% of nitrogen, employing an anaerobic system (Microbiology Anaerocult A, MERCK, Darmstadt, Germany).

After 48 h from the incubated growth media, the inhibition zones were read and marked, then the specimens for 7 and 21 days testing were transferred onto fresh spread growth media, as described above, and incubated under the same conditions. After 7 days, the inhibition zones were read and marked again, and the specimens for 21 day testing were transferred onto fresh spread growth media and incubated in the anaerobic pot until the 21st day, when the last measurements were made.

The inhibition zone was determined by measuring its diameter (mm). The size of the inhibition zone included the diameter of the specimen (4 mm). The measurements for each specimen were done in two perpendicular directions, and the mean values calculated. Integer values in millimeters were read, and in cases when the width of the inhibition zone was between two integers, the smaller integer value plus 0.5 mm was entered.

Statistical analysis was performed by One-way ANOVA followed by Post hoc-Tukey honest significant difference (HSD) test.

Materials and methods

Five commercial endodontic sealers were used in the study, namely RoekoSeal, Endomethasone N, N2, Apexit Plus and AH Plus. Three sets of 6 samples were prepared from each material as follows: (a) without antimicrobial agent, (b) with 2% (w/w) CPC and (c) with 2% (w/w) BC. All of the materials were tested at three time intervals, namely after 2 days, after 7 days and after 21 days of incubation.

Specimen preparation

Samples were prepared by mixing the components according to the manufacturers’ instructions. The samples with antimicrobial agents were prepared by mixing 2% by mass of the antimicrobial (CPC or BC, respectively) into the newly mixed cement. The paste was then placed in metal molds of dimensions: 6 mm (height) by 4 mm (diameter). The molds were closed by metal plates on both sides and clamped. The molds were placed in an incubator at 37 °C for 24 h and the specimens allowed to set.

Microbiological analysis

The agar diffusion method was used for the determination of the inhibitory effect of the specimens on 3 different test strains: S. mutans – ATCC 25175, L. casei – ATCC 4646 and A. viscosus – ATCC 19246 (manufactured by American Type Culture Collection, Manassas, VA, USA).

The bacterial strains were inoculated into BHIB (Brain Heart Infusion Broth, Oxoid, Wesel, Germany) and incubated in an anaerobic atmosphere at 37 °C for 48 h. From the bacteria grown in the liquid medium, the density of the inoculum was set to be equivalent to the McFarland 2 standard. Then, in previously marked Shaedler agar with addition of sheep blood (Schaedler + SB, Oxoid, Wesel, Germany), 350 μL of the bacterial suspension were inoculated.

After the inoculation, and a subsequent period of 15 min for absorption of the bacterial suspension by the agar plate; sterile plastic tubes were used to create wells (3 mm depth and 3.5 mm width) into the agar plate. A total of 7 wells were prepared on each dish, 6 along the perimeter and one in the center. The distance between the wells was 30 mm, and the distance between the wells and the dish wall was 15 mm. Then, the specimens were carefully placed in the wells with sterile tweezers, so that they would have close contact with the agar. The Petri dishes were left for 30 min at room temperature, and incubated anaerobically at 37 °C for 48 h. The anaerobic atmosphere for cultivation was produced by anaerobic pots, with an atmosphere of 10% of carbon dioxide and 90% of nitrogen, employing an anaerobic system (Microbiology Anaerocult A, MERCK, Darmstadt, Germany).

After 48 h from the incubated growth media, the inhibition zones were read and marked, then the specimens for 7 and 21 days testing were transferred onto fresh spread growth media, as described above, and incubated under the same conditions. After 7 days, the inhibition zones were read and marked again, and the specimens for 21 day testing were transferred onto fresh spread growth media and incubated in the anaerobic pot until the 21st day, when the last measurements were made.

The inhibition zone was determined by measuring its diameter (mm). The size of the inhibition zone included the diameter of the specimen (4 mm). The measurements for each specimen were done in two perpendicular directions, and the mean values calculated. Integer values in millimeters were read, and in cases when the width of the inhibition zone was between two integers, the smaller integer value plus 0.5 mm was entered.

Statistical analysis was performed by One-way ANOVA followed by Post hoc-Tukey honest significant difference (HSD) test.

Results

Fig. 1 shows the inhibition zone after removal of the specimen. At 2 days, the highest antimicrobial effect on S. mutans ( Graph 1 ) was shown by N2. Of the additives, BC had greater antimicrobial effect on S. mutans than CPC. Generally, there was a gradual decline in the inhibitory levels over time. There were small inhibition zones in Apexit Plus, AH Plus and RoekoSeal after 2 days without the addition of antimicrobial agent and none after 7 and 21 days; however the inhibition zones were larger when antimicrobials were added.

Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Incorporation of antimicrobial agents can be used to enhance the antibacterial effect of endodontic sealers
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