Baccor, in vitro formation of biofilm in broken endodontic files in radicular canals

The aim of this study to evaluate the biofilm formation of sulfate-reducing bacteria from two microbial species, Desulfovibrio desulfuricans (oral and environmental strain) and Desulfovibrio fairfieldensis, in root canals with fractured endodontic files in vitro and the biocorrosive changes that these strains are capable of promoting on the metal surface of endodontic files. Fourteen teeth were included with fractured # 90 Kerr files and inoculants of Desulfovibrio desulfuricans strains and Desulfovibrio fairfieldensis in a modified Postgate E culture medium. The inoculated teeth were evaluated at 28, 41, 51 and 477 days. The biofilm was evaluated through scanning electron microscopy (SEM) and confocal laser scanning microscope with the fluorophore the Live / Dead® kit. For the 477 day, chemical pickling followed, with subsequent evaluation of the metallic surface of endodontic files in the SEM. In the biofilm analyzes of 28, 51 and 477 days, a mixed biofilm was observed, with a predominance of living cells and areas of corrosion along the entire metallic surface of the file for the last time, contrasting the metallic surface of the control groups. The SRB showed cellular activity both on the metal surface of the file and on the dentin surface of the root canal at the deepest levels of the root canal, apical and medium, promoting biocorrosion along the metal surface of endodontic files.


Introduction
Different techniques are used in the removal of fractured endodontic instruments in root canals, such as hemostatic forceps for apprehending the fragment, overtaking the fragment for subsequent traction with Kerr files, ultrasound and even the use of cyanoacrylate for adhesion of the fragment with another instrument. in an attempt to achieve success in removing the lime fragment (Gaffney, et al., 1981, Coutinho, et al., 1998, Nehme, 1999, De Oliveira, 2003, Suter, et al., 2005. However, no technique is completely safe, which can lead to perforations, false canals, destruction of the tooth root or a reduction in tooth root resistance (Bahcall, et al., 2005, Terauchi, et al., 2006. In order to assist in the removal of fractured instruments from the inside of the root canal, BACCOR was developed. A biopharmaceutical based on the action of Sulfate-Reducing Bacteria (SRB) with the power to promote biocorrosion in manual endodontic files (Heggendorn, et al., 2015).
Biocorrosion or microbiologically influenced corrosion (MIC) is a type of corrosion in which microorganisms actively participate in this process, initiating or accelerating the electrochemical reaction of metallic dissolution. A fundamental step in this process is the capacity of biofilm formation by microorganisms, where, through their metabolism, they induce and maintain the oxygen concentration gradient, accelerating the corrosion process through the reduction of oxygen and the release of their metabolic products (Videla, 2003, Larry & Hamilton, 2007. In this context, it is essential to evaluate the in vitro action of BACCOR in the conditions closest to the clinical conditions to be applied, with endodontic files fractured in root canals. Therefore, the present study aimed To evaluate the biofilm formation of sulfate-reducing bacteria from two microbial species, Desulfovibrio desulfuricans (oral and environmental strain) and Desulfovibrio fairfieldensis, in root canals with fractured endodontic files in vitro and the biocorrosive changes that these strains are capable of promoting on the metal surface of endodontic files through the use of fluorescence microscopy and scanning electron microscopy techniques.

Methodology
The study was approved by the Research Ethics Committee of the Federal University of Rio de Janeiro through the consubstantiated opinion of CEP: CAAE 01500258000-09.
The teeth were removed from the 10% formaldehyde solution, washed and dipped in distilled water. After drying, the teeth were accessed in the coronary region and instrumented in the apical third (3 mm apical) (Step-back): Basic dilation to the apical limit with a file that adjusted to the anatomical diameter of the apical region, followed successive dilations 1 mm below the apex, limited by the diameter of the root canal; Scheduling, with a programmed 1-millimetre indentation for the use of a file with a higher caliber than the file previously used, with only the apical third (3 mm) being prepared; Expansion of the middle third with a wide drill no. 3 and instrumentation with a Kerr file n.º 80 and check whether the expansion achieved in the previous step was sufficient to adopt a Kerr file type 90 or 100 in the middle third (K-File colorinox®; Dentsply Maillefer; REF A 012D 031 902 00; Lot: 1306001790). The Kerr type 90 endodontic files were then rotated clockwise with apical pressure in the middle third of the root canal. Once the endodontic file had been jammed in the root canal, the middle part of the endodontic file was sectioned. Subsequently, they were adapted in a 2 ml Eppendorf microtube, sealed and autoclaved for 15 minutes. After sterilization, a Postgate E culture medium was added to the microtube in the outer apical region of the root, plus Agar-agar, 15g / l., in order to fix the tooth inside the microtube after solidification of the medium and indicate bacterial confinement within the root canal, since, in case of contamination of external regions of the root, the culture medium will indicate this contamination through the change of colour and formation of iron sulfide (Figure 1). Previously the inoculation of BACCOR, the washing of innocuous bacteria was carried out, centrifuged at 10,000 g / 05 min. for the recovery of bacterial cells, discarding the supernatant. Subsequently, the precipitate from each sample was resuspended with Postgate E culture medium without Agar-agar. A 1.0 mL aliquot of each bacterial inoculum was removed to determine the cell concentration using the most likely number (MLN) technique and then follow the inoculation of 20 µl of each inoculum into the root canal in the respective previously prepared teeth and divided according to Table 1, incubated at 30 °C in an oven, for later observation in the SEM. After the incubation period of each group (Table 1), the formation of iron sulfide in the external region of the tooth root resulting from bacterial growth in the Postgate E culture medium was evaluated, plus Agar-agar, located in the external region of the radicular tooth portion.

D13
Oral Desulfovibrio desulfuricans D14 Desulfovibrio fairfieldensis D15 New endodontic file, without specific treatment MEV Source: From the authors.

Biofilm fixation
After the incubation period, the teeth with the inoculum, followed for the fixation of the biological material, the groups were kept for 4 hs in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) prepared with Milli-Q. After 4 h, they were removed from this solution and washed three times in 0.1M cacodylate buffer, each washing time being 15 minutes. Then they were dehydrated in an increasing series of 15 minutes each, in 30, 50, 70 and 100% alcohol. Finally, the essential drying point of the material was carried out in critical point equipment (Leica Microsystems; Leica EM CPDO30). The purpose of this procedure was to replace all residual biofilm liquid with liquid CO2, which at 31 °C and 73 atm of pressure quickly convert to gaseous CO2. After the drying time, the teeth were included in acrylic resin and sectioned longitudinally with a carborundum disc with the aid of a micro grinder (Black & Decker, RT 650) in order to induce a longitudinal fracture of the tooth without reaching the conduit root region, thus forming two longitudinal areas and releasing the endodontic file from the conduit region without affecting the structure of the biofilm. Subsequently, the teeth inoculated with the files were separated into groups according to Table 1. In groups, D1, D2, D3 and D4, the root faces with root canal and endodontic files were metallized with subsequent sputtering coated in gold (15 nm thick) and examined for bacterial biofilm and surface changes using scanning electron microscopy (MEV-FEI-Inspect-S50) ( Table 1).
To analyze the possible biofilm formed on the endodontic files and in the root canal, the laser scanning confocal Research, Society and Development, v. 11, n. 1, e55511123849, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i1.23849 5 After 477 cultivation, the endodontic files of groups D10-D15 were observed, using the same protocol for observing cell viability in the MCVL with the fluorophore kit Live / Dead®. Subsequently, the endodontic files were subjected to the chemical pickling process to remove corrosion products and impurities present on the metallic surface, thus facilitating the observation of possible Pitts and/or corrosion areas. The specimens were immersed for 20 min. at 60 °C in 10% Nitric Acid solution (HNO3), washed with 70 alcohol (hydrated ethyl alcohol 70° INPM) and acetone and dried with hot air. Then the groups were observed in the SEM.

Results
The cell concentration obtained by the MLN technique in the inoculants of the biocorrosive assay in the teeth in vitro is shown in Table 2. After the 41-day incubation period, each group was assessed macroscopically with the interruption of the biocorrosion assay. Group D1, the tooth with inoculation of oral Desulfovibrio desulfuricans, showed darkening in the cementum near the apical foramen ( Figure 2). However, after the critical point procedure of the material, this darkening of the apical region was no longer present. The other groups did not present any colour changes or other types of abnormalities in the coronary or radicular tooth portions. Source: From the authors.

Scanning electron microscopy
All images presented a smear layer of different intensities, making it challenging to identify biofilms or bacteria.
However, group D1 revealed an area suggestive of biofilm on the surface of the endodontic file ( Figure 3D Figures 3E.1, 3E.2), which may indicate that the smear-layer impaired, hiding, a large part of the bacteria. It was also possible to verify the suggestive presence of bacteria in other fields, with similar measurements between the different areas ( Figures 3F, 3G). However, such images suggested a deposit on the cells, which made visualization difficult. On the surface of the endodontic file in group D2, the images suggested the presence of EPS and bacteria in figures 3H, 3I and 3J, in addition to visualizing the rupture of the biofilm, demonstrating the metallic structure of the file below that point ( Figures 3L.1 and 3L.2). Source: From the authors.

Biofilm Analysis Association -MCVL and SEM
In group D5, with 28 days of biofilm formation, it was possible to visualize a mixed biofilm with dead and living cells  Source: From the authors.
The control groups showed no difference between their treatments, with the presence of only culture medium, Group D7, and without any type of inoculum, Group D6. However, in both control groups, a natural smear layer fluorescence was observed when the laser was applied, both on the dentin surface, root canal, and on the endodontic file, between 488 and 529 nm (Excitation Length and Emission Length) of λ of wave, which covers the same emission / excitation frequency of the SYTO® 9 fluorophore, but with a lower fluorescence intensity when compared to samples with active biofilm. The SEM analysis showed a strong presence of amorphous crystals, compatible with smear layer on the dentin surface (

Association of Biofilm Analysis with Corrosion Areas -MCVL and SEM
After 477

Discussion
The process of bacterial adhesion and biofilm formation and its morphology can vary depending on the species, the surface composition, and environmental factors (Clark, et al., 2007, Trinidad, et al., 2010. Therefore, it the important of trying to analyze the closest possible conditions to BACCOR in vitro. Regarding the difference in surface composition, SRB were able to form biofilm both on the metallic surface and on the inner surface of the root canal. The data suggested an association of smear layer with the formation of SRB biofilm, leading to the belief that the first can support bacterial adhesion, both on the dentin surface and the metal surface of the endodontic file. Regarding time, SRB remained viable at all times used for 28, 41, 51 and 477 days, in addition to presenting bacterial adhesion on the metal surface of the endodontic file in the shortest incubation time, 28 days. In addition to proving the formation of MIC by SRB, this study demonstrates that SRB remains viable within the root canal for a long period. Previously, the presence of SRB in the oral cavity, on dentinal surfaces, in saliva had already been identified (Heggendorn, et al., 2013 and. Therefore, in addition to making it possible to prove SRB biocorrosion in which can be related to sample preparation. Clark et al. (2007) detected in the SEM the presence of numerous filaments that linked the SRB to each other in the biofilm, similar to those found in our samples (Figures 3 and 4). The authors argued that such protein filaments are essential structures in the maturation of the SRB biofilm, indicating that Desulfovibrio Vulgaris is based more on protein filaments than on the exopolysaccharide matrix for the fixation and initial formation of the biofilm (Clark, et al., 2007). Later, Remoundaki et al. (2008) stated that solid structures in the form of rods could be bacterial cells encapsulated by zinc and iron sulfides. The authors described through the SEM-EDX a SRB encapsulation, shaped like a 10µm rod, by a cloudy fog corresponding to the deposition of metallic sulfides on the surface of the cell wall and or the area adjacent to the bacterial cell (Remoundaki et al., 2008). Based on the findings of this author, during the analysis of the images obtained in the SEM, morphological differences were identified between the SRB cells, between different samples and in the same sample, with different sizes being identified between the cells (unpublished data).
The process of biocorrosion of iron by SRB must be seen as a phenomenon of rupture of the passive film of steel by the corrosive metabolites that SRB release in the environment. This process can be intensified by the presence of chloride ions, associated with biogenic sulfides, released by SRB, causing synergy and an increase in the speed of attack on the metal (Videla, 2003, Larry & Hamilton 2007. The corrosion caused by SRB is predominantly located, predominantly by pirates, with corrosion products with little adherence, dark coloring, and a characteristic odor of hydrogen sulfide (Videla, 2003, Lopes, et al., 2006. This biocorrosive process would be desirable in endodontic files fractures within the root canals since they would facilitate the detachment of the endodontic file from the root canal walls. Therefore, the proof of cell viability of SRB in all inoculated SRB groups, both on the metal surface of the file, suggesting the possibility of MIC, as well as on the root canal wall, in the apical region and the middle third of the root canal, indicates that the SRB would not be immobilized only in the access area of the inoculum, but managed to pass through the fractured endodontic file barrier, reaching apical regions of the root canal as well as the active tip of the fractured endodontic file in the root canal.
In previous analyzes, the biocorrosion power of BACCOR was proven, with strains of D. desulfuricas and D.
fairfieldensis, in Kerr-type endodontic files embedded in cross-sectioned acrylic resin with 28 days of incubation. The pattern of corrosion in the work of Heggendorn, et al. (2015) was similar to those presented in this work: in areas of the interface (edge) between files and embedded resin (Heggendorn, et al., 2015) that would correspond to the edge areas of cutting endodontic files embedded in teeth. It was also noticed in areas of pre-existing structural defects where the biocorrosive process was able to act (Heggendorn, et al., 2015), sealing the cutting edges with pullout where the area of corrosion was identified in group D12.
Control samples D10 and D11 suggested areas of abiotic corrosion without the presence of SRB. Such a phenomenon arises from the exposure of the culture medium in the endodontic file (Group D10) in the root canal; the culture medium alone would promote abiotic corrosion. The group D11, without any treatment, only the exposure of the endodontic file in the root canal was able to encourage corrosion in some areas, such fact can be related to residual moisture in the dentine, previously treated with sodium hypochlorite and even by the simple presence of oxygen, even if in reduced concentration. However, when comparing the control groups D10 and D11 with the groups inoculated with SRB (D12, D13 and D14), the biocorrosion provided by SRB were able to act to a great extent, suggesting a more aggressive attack on the metallic surface when compared to abiotic corrosion in the control samples D10 and D11, were part of the surface was shown to be integral. Trinidad et al. (2010) recommend a combination of several techniques when investigating biofilms. The increase in knowledge about biofilms is based on the scanning electron microscope and the scanning laser confocal microscopy, which complement each other in the knowledge of the biofilm ultrastructure and the cell viability in the development of biofilm (Trinidad, et al., 2010). In fact, the application of these two methodologies led to analyzes that, if applied in isolation, the conclusions presented in this work would not be reached. MCVL and SEM made it possible to check and analyze the cellular activity of the SRB and the biofilm structure, respectively, for periods of up to 28, 41, 58 and 477 days of cultivation throughout the entire root canal with the fractured endodontic file in its interior.

Conclusion
The SRB showed cellular activity both on the metal surface of the file and on the dentin surface of the root canal at the deepest levels of the root canal, apical and medium, promoting biocorrosion along the metal surface of endodontic files.
This corrosion was shown to be diffuse along the surface of the endodontic file, also showing areas of corrosion on the cutting edges of the instrument, which would be what is desired to facilitate the detachment of the instrument when fractured inside the root canal. In parallel, the SRB were able to form biofilm on the dentinal surface of the root canals, with no changes observed on these surfaces. .