Abstract
Objective
Bacteria persisting in the root canal system may thwart endodontic therapy. It is therefore interesting to know whether clinically available root canal sealers have an antimicrobial effect. The objective of the present in vitro study was to investigate the antibacterial effect of various sealers on the endodontologically detectable species Enterococcus faecalis ( E. faecalis ), Fusobacterium nucleatum ( F. nucleatum ) and Porphyromonas gingivalis ( P. gingivalis ).
Methods
The antibacterial effectiveness of the sealers was tested by means of the agar diffusion test (ADT) and the direct contact test (DCT). Eight different sealers (AH Plus ® , Hermetic ® , RoekoSeal ® , Sealapex ® , Apexit Plus ® , 2Seal ® , EndoREZ ® and ProRoot MTA ® ) and two temporary sealers (Calxyl ® and Gangraena Merz ® ) were tested.
At first, 100 μl of bacterial suspension (BS) of each individual micro-organism (optical density (OD) 0.5) was applied separately to Schaedler agar plates for the ADT. Subsequently, freshly mixed and set sealer was applied. After 48 h of incubation, the inhibition zones were measured.
Further, 18 mg of sealer were put into each well of 48-well cell culture plates and overlaid with 400 μl of Schaedler liquid medium and 100 μl of BS (OD 0.5) in monoculture. Bacterial growth was determined by the DCT from the optical density of the liquid by photospectrometry after 2, 4, 6, 8, 10, 12 and 24 h.
Results
For the application, the sealer Hermetic ® , a significant suppression of the species E. faecalis , F. nucleatum and P. gingivalis was detected in both the ADT and the DCT. AH Plus ® showed a suppressive effect on E. faecalis and F. nucleatum in the DCT. With all other sealers tested, E. faecalis was not suppressible.
RoekoSeal ® , Calxyl ® and Gangraena-Merz ® showed no antibacterial effect on the tested species whatsoever.
Significance
We have shown in both ADT and DCT that some root canal sealers suppress the growth of E. faecalis in vitro.
1
Introduction
Currently, dental materials are frequently required not only to be biocompatible but also to have an antibacterial effect .
The materials are expected to have low cytotoxicity on the one hand, and to prevent bacterial colonization and formation of a biofilm on the material boundary surface on the other. Antibacterial materials are desired to be able to suppress bacteria and inhibit the development of bacterial biofilms as far as possible .
At present, suppression of oral species is attempted by various strategies, some of which are in clinical application already . Within the scope of the filling therapy, for example, there already exist adhesive systems with antibacterial effect ; composites with antibacterial effect are also known .
Bacteria especially persisting in the endodontium may jeopardize the success of the therapy even after painstaking mechanical preparation and disinfection of the root canals .
The spectrum of the bacteria detected in the primarily or secondarily infected root canal may be relatively broad . In most cases, it is the endodontopathogenic species Enterococcus faecalis that is a key pathogenic factor in treatment failure . This species is detectable in about 77% of the cases resistant to treatment .
The species Fusobacterium nucleatum and Porphyromonas gingivalis , both being known parodontopathogens, are frequently observed in connection with apical periodontitis . F. nucleatum was detected in 64% of the infected root canals . In another study, P . gingivalis occurred in 48% of the endodontic samples . Obviously, an antibacterial effect of the root filling materials would be desirable, especially from the clinical point of view.
Generally, at the end of every endodontic therapy, one tries to seal the root canal so that it is as bacteria-proof as possible. After mechanical preparation, the root canal is obturated with a (semi-)solid material and a root canal sealer . Commercial sealers presently available differ in their chemical composition as well as in their physico-chemical properties . As a rule, the sealer is offered as a binary preparation consisting either of two pastes or of a powder and a liquid. The sealer, blended into a paste, is filled into the root canal together with the core filling material. After the setting process, the sealer fulfills the purpose of sealing the root canal .
The issue of an adjuvant antibacterial effect of the sealers is becoming increasingly important, especially from the clinical viewpoint, because it is not uncommon that, e.g., E. faecalis persists in the dentinal tubules after treatment, causing reinfection .
So far, the sealers currently on the market have hardly been studied with regard to their antibacterial effect, although it is a demand expressed also by the European Society of Endodontology (ESE) that sealers should, in addition to completely obturating the root canal, preferably have a nonsupportive effect on bacterial growth . Thus it is of interest, especially to the clinician, to know the antimicrobial effects that can be expected from currently available sealers.
Therefore, it was the aim of this in vitro study to test currently available sealers for their antibacterial effect on endopathogenic species.
2
Materials and methods
2.1
Sealer samples
In this in vitro study, we tested eight definitive root canal sealers for their antibacterial effectiveness. Depending on their basic components, the sealers were assigned to eight groups ( Table A1 ).
We further included two temporary root canal cements containing calcium hydroxide (Calxyl Rot ® und Gangraena-Merz ® ) and assigned them to group 9 ( Table A1 ).
Each of the sealers was mixed under sterile conditions and in accordance with the instructions of the respective manufacturers. The freshly mixed sealers were subjected to the agar diffusion test (ADT) and the direct contact test (DCT).
In addition, the sealers were also subjected to the ADT after setting. To make samples of the set sealer samples, sealer was spread on cellulose plates (diameter 9 mm) and stored for six days in an incubator for setting at 37 °C and 90% humidity. In case of EndoREZ ® , in addition, light curing of the surface was applied for 40 s with a polymerization lamp (bluephase C8 ® , Ivoclar Vivadent, Schaan, Lichtenstein) immediately after mixing and spreading on the cellulose plates.
2.2
Bacterial species
The following species were used for investigating the antibacterial effect of the sealers: the Gram-positive, facultatively anaerobic species E. faecalis (DSMZ 20376), and the Gram-negative anaerobic species F. nucleatum (DSMZ 20482) and P. gingivalis (DSMZ 20709). The bacteria were cultivated in monocultures in 10 ml Schaedler liquid medium (Oxoid Ltd, Hampshire, UK) under anaerobic standard conditions for 24 h.
2.3
Agar diffusion test (ADT)
By means of the agar diffusion test we determined both the antibacterial effectiveness of each of the sealers in freshly mixed condition and after setting.
For each sealer, 100 μl of bacterial suspension (optical density OD 0.5) in monoculture was applied to Schaedler agar (Schaedler Anaerobe agar, Oxoid Ltd, Hampshire, UK; Vitamin K (Konakion ® ) 0.1%; sheep’s blood 6%).
20 mg of each freshly mixed sealer were applied to sterile cellulose plates of 9 mm diameter and spread evenly over the entire disk surface.
The cellulose plates so prepared were put – sealer-spread face down – onto the agar plates prepared with bacterial suspension.
The antibacterial effects of the freshly mixed sealers were studied in ten test series.
In a second experiment, the sealers were tested in the set state. For this, the samples were placed on the agar plates prepared with bacterial suspension, with the sealer having direct contact with the agar plates. Six test series were made per sealer.
In both experiments, then, the agar plates were cultivated for 48 h under anaerobic conditions at 37 °C. The inhibition zones produced were measured with a ruler with an accuracy of 1 mm. Afterwards mean values were formed of the measured inhibition zones according to the results of the test groups.
In both experiments, chlorhexidine (Chlorhexamed forte ® 0.2%, GlaxoSmithKline Consumer Healthcare, Bühl, Germany) was used as a positive, and distilled water as a negative control.
2.4
Direct contact test (DCT)
In the DCT, the sealers that had shown a good antibacterial effect in the ADT were investigated in more detail. Here, we included only those sealers which had, in the ADT test, inhibited the growth of at least two species in the freshly mixed state and at least one species in the set state.
The sealers were applied to the wells of 48-well cell culture plates by means of a circular punch of 7 mm diameter. In that way, about 18 mg of sealer was placed on the bottom of each well. The sealers were then covered with 400 μl Schaedler liquid medium (Oxoid Ltd, Hampshire, UK), and 100 μl of bacterial suspension of 0.5 optical density was added per well.
The culture medium was used as a negative control, whereas 100 μl of bacterial suspension with baseline cell concentration, mixed with 400 μl of Schaedler liquid medium, was used as a positive control.
The plates thus prepared were incubated in an anaerobic environment at 37 °C. In triple determination, we took 3 × 100 μl of eluate of each test substance at seven test times, after 0, 2, 4, 6, 8, 12 and 24 h, and subsequently determined the optical density at 560 nm. As a result, we had altogether 9 readings for each sealer at each of the various time points.
The bacterial growth correlates with the optical density of the suspension; i.e., as the bacteria multiply, the opacity of the liquid increases and, in comparison with the control sample, yields information on the antibacterial effect of the substances tested .
To determine the bactericidal effect of the tested materials, dilution series of the eluate-bacterial suspension were performed after an incubation time of 24 h; three times we applied 20 μl each of the respective dilution series to Schaedler agar (Schaedler Anaerobe agar, Oxoid Ltd, Hampshire, UK; Vitamin K (Konakion ® ) 0.1%; sheep’s blood 6%). The agar plates thus prepared were incubated for 72 h under anaerobic conditions at 37 °C, and the colony-forming units (CFU) were determined by counting.
2.5
Statistical analysis
The results of this study were statistically analyzed using SPSS statistical software (IBM; version 19.0).
A significant difference between the various groups in the ADT was determined with the Mann–Whitney U -test. Significant differences between the groups in the DCT were identified by multiple comparisons according to Bonferroni and, in case of the CFUs, by means of a two-tailed t -test for independent random samples. The significance level was p < 0.05.
2
Materials and methods
2.1
Sealer samples
In this in vitro study, we tested eight definitive root canal sealers for their antibacterial effectiveness. Depending on their basic components, the sealers were assigned to eight groups ( Table A1 ).
We further included two temporary root canal cements containing calcium hydroxide (Calxyl Rot ® und Gangraena-Merz ® ) and assigned them to group 9 ( Table A1 ).
Each of the sealers was mixed under sterile conditions and in accordance with the instructions of the respective manufacturers. The freshly mixed sealers were subjected to the agar diffusion test (ADT) and the direct contact test (DCT).
In addition, the sealers were also subjected to the ADT after setting. To make samples of the set sealer samples, sealer was spread on cellulose plates (diameter 9 mm) and stored for six days in an incubator for setting at 37 °C and 90% humidity. In case of EndoREZ ® , in addition, light curing of the surface was applied for 40 s with a polymerization lamp (bluephase C8 ® , Ivoclar Vivadent, Schaan, Lichtenstein) immediately after mixing and spreading on the cellulose plates.
2.2
Bacterial species
The following species were used for investigating the antibacterial effect of the sealers: the Gram-positive, facultatively anaerobic species E. faecalis (DSMZ 20376), and the Gram-negative anaerobic species F. nucleatum (DSMZ 20482) and P. gingivalis (DSMZ 20709). The bacteria were cultivated in monocultures in 10 ml Schaedler liquid medium (Oxoid Ltd, Hampshire, UK) under anaerobic standard conditions for 24 h.
2.3
Agar diffusion test (ADT)
By means of the agar diffusion test we determined both the antibacterial effectiveness of each of the sealers in freshly mixed condition and after setting.
For each sealer, 100 μl of bacterial suspension (optical density OD 0.5) in monoculture was applied to Schaedler agar (Schaedler Anaerobe agar, Oxoid Ltd, Hampshire, UK; Vitamin K (Konakion ® ) 0.1%; sheep’s blood 6%).
20 mg of each freshly mixed sealer were applied to sterile cellulose plates of 9 mm diameter and spread evenly over the entire disk surface.
The cellulose plates so prepared were put – sealer-spread face down – onto the agar plates prepared with bacterial suspension.
The antibacterial effects of the freshly mixed sealers were studied in ten test series.
In a second experiment, the sealers were tested in the set state. For this, the samples were placed on the agar plates prepared with bacterial suspension, with the sealer having direct contact with the agar plates. Six test series were made per sealer.
In both experiments, then, the agar plates were cultivated for 48 h under anaerobic conditions at 37 °C. The inhibition zones produced were measured with a ruler with an accuracy of 1 mm. Afterwards mean values were formed of the measured inhibition zones according to the results of the test groups.
In both experiments, chlorhexidine (Chlorhexamed forte ® 0.2%, GlaxoSmithKline Consumer Healthcare, Bühl, Germany) was used as a positive, and distilled water as a negative control.
2.4
Direct contact test (DCT)
In the DCT, the sealers that had shown a good antibacterial effect in the ADT were investigated in more detail. Here, we included only those sealers which had, in the ADT test, inhibited the growth of at least two species in the freshly mixed state and at least one species in the set state.
The sealers were applied to the wells of 48-well cell culture plates by means of a circular punch of 7 mm diameter. In that way, about 18 mg of sealer was placed on the bottom of each well. The sealers were then covered with 400 μl Schaedler liquid medium (Oxoid Ltd, Hampshire, UK), and 100 μl of bacterial suspension of 0.5 optical density was added per well.
The culture medium was used as a negative control, whereas 100 μl of bacterial suspension with baseline cell concentration, mixed with 400 μl of Schaedler liquid medium, was used as a positive control.
The plates thus prepared were incubated in an anaerobic environment at 37 °C. In triple determination, we took 3 × 100 μl of eluate of each test substance at seven test times, after 0, 2, 4, 6, 8, 12 and 24 h, and subsequently determined the optical density at 560 nm. As a result, we had altogether 9 readings for each sealer at each of the various time points.
The bacterial growth correlates with the optical density of the suspension; i.e., as the bacteria multiply, the opacity of the liquid increases and, in comparison with the control sample, yields information on the antibacterial effect of the substances tested .
To determine the bactericidal effect of the tested materials, dilution series of the eluate-bacterial suspension were performed after an incubation time of 24 h; three times we applied 20 μl each of the respective dilution series to Schaedler agar (Schaedler Anaerobe agar, Oxoid Ltd, Hampshire, UK; Vitamin K (Konakion ® ) 0.1%; sheep’s blood 6%). The agar plates thus prepared were incubated for 72 h under anaerobic conditions at 37 °C, and the colony-forming units (CFU) were determined by counting.
2.5
Statistical analysis
The results of this study were statistically analyzed using SPSS statistical software (IBM; version 19.0).
A significant difference between the various groups in the ADT was determined with the Mann–Whitney U -test. Significant differences between the groups in the DCT were identified by multiple comparisons according to Bonferroni and, in case of the CFUs, by means of a two-tailed t -test for independent random samples. The significance level was p < 0.05.
3
Results
In this in vitro study, the antibacterial effect of eight root canal sealers and the temporarily used materials Calxyl ® and Gangraena-Merz ® on the species E. faecalis , F. nucleatum and P. gingivalis was tested. In each case, both the freshly mixed and the set sealer were employed.
3.1
Agar diffusion test (ADT)
In the ADT, only the Hermetic ® sealer in the freshly mixed state markedly suppressed the growth of E. faecalis ( Fig. 1 ).
In the freshly mixed state AH Plus ® , Hermetic ® , Sealapex ® , 2Seal ® EndoREZ ® and ProRoot MTA ® showed a suppressive effect on F. nucleatum . After hardening, inhibition of F. nucleatum could only be observed for Hermetic ® , Sealapex ® and EndoREZ ® ( Fig. 2 ).
