Background

Bacteria play an essential role in initiation, progression, and persistence of apical periodontitis (Möller et al. 1981; Kakehashi et al. 1969). In the last decades, it has been shown that intracanal bacteria are organized in a biofilm which enables higher resistance to both antimicrobial agents and host defense mechanisms (Gilbert et al. 1997; Costerton et al. 1994; Costerton et al. 1987). Complete elimination of the biofilm from an infected root canal is usually not achievable by applying the common techniques (Fransson et al. 2013; Sadık et al. 2013; Cardoso et al. 2008; Huth et al. 2009; Hülsmann et al. 2003; Vianna et al. 2006; Nair et al. 2005; Peters et al. 2010; Peters 2004). Mechanical preparation is unable to reach all root canal walls (Peters et al. 2010; Peters 2004). The various irrigation protocols, including agents such as NaOCl (Nair et al. 2005), CHX (Vianna et al. 2006), EDTA (Hülsmann et al. 2003), the usage of Ozone (Cardoso et al. 2008; Huth et al. 2009), and direct and indirect laser irradiation (Fransson et al. 2013; Sadık et al. 2013) were also unable to achieve a sterile canal.

A common reason for failure of root canal treatment is residual bacterial biofilm or reinfection due to coronal seal inadequacy (Nair 2014), (Ray and Trope 1995).

Recently, the idea of using macromolecules with antibacterial features has evolved (Kenawy et al. 2007; Barros et al. 2014; Kesler Shvero et al. 2013; Shrestha et al. 2010; Kishen et al. 2008). The addition of nanoparticle macromolecules to sealers gained antibiofilm properties as shown in several in vitro studies (Barros et al. 2014; Kesler Shvero et al. 2013).

A new concept is suggested by adding of non-nanoparticle macromolecules with antibiofilm features (Biosafe®, HM4100, BIOSAFE Inc., Pittsburg, PA, USA) to epoxy sealer. BioSafe® has received FDA approval (Application number 292 from December 12th 2013) and is based on a Silane quaternary ammonium salt. It has already been used as an additive to plastic devices such as catheters and keypad covers in order to prevent biofilm formation (D'Antonio et al. 2013).

The aim of the present study was to test the in vitro antibacterial effect of epoxy sealer BJM ROOT CANAL SEALER® (BJM Laboratories Ltd., Or-Yehuda, Israel), incorporated with non-nanoparticle macromolecules (BioSafe®, HM4100, BIOSAFE Inc., Pittsburg, PA, USA) against existing biofilm of Enterococcus faecalis and its ability to inhibit de-novo biofilm formation of Enterococcus faecalis.

Methods

Tested materials preparation

The tests specimen discs (6 mm diameter and 3 mm thickness) were prepared utilizing a split Silicon molds. Prior to the specimen’s preparation, Vaseline was applied over the mold to prevent dripping and to ease sample removal. The sealer was syringed directly from the auto-mix syringe into the mold cavities utilizing a plastic mixer. The surfaces of the discs were flat and even. The discs were kept for 24 h in a dry oven (laboratory incubator) constantly maintained at 37 °C to allow setting. Afterwards, the discs were emerged in water for 24 h at the oven (laboratory incubator) constantly maintained at 37 °C.

Six discs of epoxy sealer (BJM) incorporated with various concentrations of immobilized quaternary ammonium particles (0.4, 0.8, 1.6, 3.3% w/v) or without any addition (as negative control) were prepared and sterilized by autoclaving.

Bacteria and growth conditions: Enterococcus faecalis (ATCC 51299) was subcultured for 48 h in brain heart infusion (BHI) broth (Difco) at 37 °C under aerobic conditions.

Biofilm formation assay

Tested material discs were placed at the bottom of a 96-well microplate. Discs were inoculated with 20 μL of the tested bacterial suspensions (1.0 OD, 600 nm) and allowed to adhere for 3 min at room temperature. Following bacterial adherence, the wells were gently filled with 200 μL of growth media (BHI broth) and incubated for 48 h at 37 °C under aerobic conditions to allow biofilm formation.

Following incubation, the supernatant in each well was removed and discarded. Biofilms formed on the tested discs were stained using 200 μL crystal violet solution (BD Difco™BBL™, 0.1% w/v) for 15 min. Excess stain was washed three times with 200 μL of phosphate-buffered saline (PBS), followed by destaining with 200 μL of acetic acid (33% v/v; 30 min). The stain eluate was tenfold diluted with PBS and quantified spectrophotometrically using a microplate reader (M.R.C, China, 600 nm(. Experiments were performed in six replicates.

Biofilm viability assay

Enterococcus faecalis (ATCC 51299) biofilms were prepared by placing 20 μL of the above tested bacterial suspension (1 OD, 600 nm) into each of 96-well microplate, followed by 3-min incubation at room temperature to allow bacterial adherence, following which wells were gently filled with 200 μL of growth medium (BHI broth). Microplates were incubated for 48 h at 37 °C under aerobic conditions to facilitate biofilm formation.

Following biofilm formation, the supernatant in each well was removed and discarded. Each well was refilled with 100 μL saline and the discs (with the tested materials) were placed on top of the grown biofilm, in direct contact for 1 h at 37 °C under aerobic conditions.

Following 1 h, the tested discs were removed and biofilms were disrupted and stained using Bacteria Live/Dead assay (PromoKine). Slides were prepared using wet mount (10 μL) of each sample and the bacteria were stained for vitality using DMAO green stain for live bacteria and EthD-III red stain for non-vital ones. The Live/Dead bacterial ratio was determined using fluorescence microscopy (X1000, L3201LED, MRC) by analyzing digital images using a manual counter of six random fields (320X320 dpi) of the slides prepared from each biofilm. Experiments were performed in six replicates.

Statistical analysis

To compare the effect of the different quaternary ammonium particles concentrations on the quantity variables, ANOVA was applied with post-hoc pairwise comparisons according to Scheffe and Dunnett. All the tests applied were two-tailed and p value ≤ 0.05 was considered statistically significant.

Results

Biofilm formation assay

The effect of quaternary ammonium particles concentration on de novo biofilm formation is presented in Fig. 1. Results demonstrated significant reductions in de-novo biofilm formation of 25 and 72% in the higher quaternary ammonium concentrations of 1.6 and 3.3% w/v respectively (p < 0.001 for both) as compared to the no-addition control discs. Smaller reductions in de-novo biofilm formation were observed at the lower quaternary ammonium particles concentrations (i.e., 0.4 and 0.8% w/v); however, these were not statistically significant (p = 0.309 and p = 0.215, respectively).

Fig. 1
figure 1

Mean results (± SD) of de-novo biofilm formation amount on the tested concentrations of the quaternary ammonium incorporated epoxy discs and the control (0, 0.4, 0.8, 1.6, and 3.3% w/v). Results are expressed as optical density (OD) at 595 nm

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Biofilm viability assay

The effect of quaternary ammonium particles concentration on biofilm viability is presented in Fig. 2. Significant reductions in biofilm viability of 20 and 36% were observed in the higher concentrations of 1.6 and 3.3% w/v respectively, as compared to the no-addition control discs (p < 0.001 for both). Lower reductions in biofilm viability were observed at the lower quaternary ammonium particles concentrations (i.e., 0.4 and 0.8% w/v); however, they were not statistically significant (p = 0.456 and p = 0.371, respectively).

Fig. 2
figure 2

Mean results (± SD) of the effect of various concentrations of quaternary ammonium incorporated epoxy discs and the control (0, 0.4, 0.8, 1.6, and 3.3% w/v) on biofilm viability. Results are expressed as Live/Dead bacterial ratio

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The effect of quaternary ammonium particles concentration on biofilm viability is also demonstrated by fluorescence microscopy imaging (Fig. 3). In the higher concentrations of 1.6 and 3.3% w/v, more dead bacteria (dyed red-orange) are observed, as opposed to more live bacteria (dyed green) in the lower concentrations of quaternary ammonium incorporated epoxy discs.

Fig. 3
figure 3

Fluorescence microscopy images of live (green) and dead (red-orange) bacteria in various concentrations of quaternary ammonium incorporated epoxy discs. a Control group (0%). b 0.4% w/v. c 0.8% w/v. d 1.6% w/v. e 3.3% w/v

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Discussion

Since 2005, some researchers define apical periodontitis as an intracanal biofilm-induced disease (Nair et al. 2005). Several strategies have been proposed in order to eliminate the residual biofilm, including different instrumental techniques (Peters et al. 2010; Peters 2004), agitation of irrigation (Gu et al. 2009), ozone treatment (Cardoso et al. 2008; Huth et al. 2009), direct laser irradiation (Fransson et al. 2013; Sadık et al. 2013), and photodynamic therapy techniques (PDT) (Schlafer et al. 2010; Siqueira and Rôças 2011; de Oliveira et al. 2014).

A new approach of insoluble macromolecular disinfectants has been introduced, which can inactivate the target microorganisms by direct contact without releasing any systemic antibacterial agents (Kenawy et al. 2007). The mechanism is based on the positive charge of the macromolecules which interacts and thus disrupts the negatively charged bacterial membrane. This approach has led to the addition of various nanoparticles, like Chitozan, Ag, and quaternary ammonium polyethyleneimine (QA-PEI), to irrigants and intracanal medications and sealers (Shrestha et al. 2010; Kishen et al. 2008; Allaker 2010; Rai et al. 2012; Wu et al. 2014; Shrestha and Kishen 2014; Chen et al. 2012; Upadya et al. 2011; Rabea et al. 2003) . Nevertheless, no technique has yet been able to completely eliminate the residual biofilm.

Another approach is preventing biofilm formation and/or survival. Experiments with synthetic nanoparticle quaternary ammonium polyethyleneimine (QA-PEI) have shown that their addition to dental materials has prevented biofilm formation on the surfaces of these dental materials (Beyth et al. 2006; Beyth et al. 2008).

One of the reasons of root canal treatment failure is lack of proper coronal sealing (Ray and Trope 1995; Keinan et al. 2011; Heling et al. 2002; Gillen et al. 2011). When the coronal restoration is compromised, a coronal leakage may occur, and the root canal system is usually subjected to bacterial infection (Keinan et al. 2011; Heling et al. 2002; Gillen et al. 2011).

A sealer with antibiofilm properties could have an advantage against residual biofilm in the root canal. The use of nonsoluble antibacterial macromolecules may provide long-term antibiofilm action.

BioSafe® (HM4100, BIOSAFE Inc., Pittsburg, PA, USA) comprises of macromolecules with quaternary ammonium groups. It has FDA approval and is used as an additive to catheters and plastic medical devices for prevention of biofilm formation.

In the present study, BioSafe® (HM4100, BIOSAFE Inc., Pittsburg, PA, USA) was added to a new epoxy resin sealer (BJM ROOT CANAL SEALER®, BJM Laboratories Ltd., Or-Yehuda, Israel). This compound qualifies ISO standard (Solomonov and Itzhak 2017). Biosafe® incorporated epoxy resin sealer was tested for its in vitro antibacterial effect against existing biofilm, and for its ability to inhibit de-novo biofilm formation. Different macromolecule concentrations were examined in order to find the most efficient one.

The results demonstrated significant reductions in biofilm viability (20 and 36%) in the higher Biosafe® concentrations (1.6 and 3.3% w/v, respectively). Hence, due to its possible influence on residual Biofilm, Biosafe® might enhance the antibacterial effect of the root canal sealer.

The microscopic fluorescence imaging technique has also confirmed the above findings, where in the higher Biosafe® concentrations (1.6 and 3.3% w/v) more dead bacteria are observed as opposed to the lower ones.

Significant reductions (25 and 72%) were also observed in de-novo biofilm formation in the higher concentrations (1.6 and 3.3% w/v respectively). This might have a potential for use in biofilm prevention. If this effect will be found to be long lasting, this could alter our perception regarding the limitations of the coronal seal, especially when post-endodontic restoration is not completed immediately after the endodontic treatment. Hence, the phenomenon of restoration leakage along the time might have much less clinical consequences.

Clearly, because this is an in-vitro model (monobiofilm and non-canal environment), future research is required in order to test the longevity of the antibiofilm effect, the physical properties of the sealer incorporated with BioSafe® (HM4100, BIOSAFE Inc., Pittsburg, PA, USA), and it is efficacy in the environment of the root canal. Surely, further research is needed to prove clinical efficacy.

Conclusions

Quaternary ammonium macromolecule incorporated in epoxy root canal sealer discs showed a pronounced reduction of de-novo biofilm formation in the higher concentrations (1.6 and 3.3% w/v), as well as some antibacterial effect against existing biofilm of Enterococcus faecalis. This may be effective for prevention of de-novo formation of bacterial biofilm in treated root canals.