Successful root canal treatment requires the elimination of pulp tissues, microbial biofilm, and bacterial byproducts from the root canal system [1]. Mechanical debridement is effective in bacterial reduction; however, due to the complexity of the canal system, such as isthmuses, ramifications, and lateral canals, 35–50% of the canal space remains uninstrumented and thereby associated with microbial remnants [2, 3, 4]. Mesial roots of mandibular molars commonly contain isthmuses in the apical 3–5 mm, with 22% having complete isthmuses and 37% having partial isthmuses [5, 6]. Bacteria or infected tissue left in these spaces can be a cause of persistent periapical lesion and root canal failure [2, 7]. The Gram-positive bacterium E. faecalis is a natural inhabitant of the gastroenterological tract of humans and is commonly associated with hospital acquired illnesses as well as endodontic infections [8]. Moreover, E. faecalis can survive root canal treatment due to its ability to invade dentinal tubules and to withstand harsh environments such as nutrient deprivation, and tolerance to disinfectants and high pH [9]. In addition, it develops resistance to antibiotics, so it has become a major threat in hospital settings [10, 11].

The chemomechanical preparation of the root canal system is the most important phase for bacterial elimination in endodontic treatment [12]. Currently, there are several file systems with different properties in relation to the type of alloy, cutting efficiency, and shaping ability. TS system (Dentsply Tulsa Dental Specialties, Tulsa, OK) consists in an S-shape design with four different tips (20, 25, 30, 40) and taper 0.06 in the apical 2 mm [13]. This unique design allows greater adaptation to the canals various shapes. According to the manufacturer, this system creates an envelope of motion that can predictably better disrupt biofilms allowing for bacterial reduction. Another instrumentation system is the VB rotary files (Dentsply Tulsa Dental Specialties, Tulsa, OK). This system contains a distinctive color with a visible titanium oxide layer, as a result of new processing of NiTi wire [14]. The manufacturers claim that VB has a 42% higher peak torque strength increase over M-Wire NiTi. VB system works in a rotational, non-reciprocating movement. Both systems tested are made of NiTi.To date, there are no ex vivo studies in the literature comparing with through quantitative analysis, the ability of mechanical instrumentation with TS and VB in bacteria removal. Thus, the purpose of our study is to compare the effectiveness, by real-time PCR, of TS and VB in removing E. faecalis from the mesial canals and isthmuses of mandibular molars.

All inoculated teeth allowed for growth and amplification of E. faecalis. There was no significant difference in the bacterial load between each sample prior to instrumentation. Absence of detectable DNA in the negative control teeth following qRT-PCR insured complete elimination of E. faecalis in the teeth canal systems prior to inoculation. The bacterial loads from teeth treated with VB or TS as determined by qRT-PCR were compared to those from the positive control teeth, which were not subjected to instrumentation or irrigation. Treatment with each of the file systems resulted in a significant decrease in bacterial load, though the reduction was greater for those teeth treated with TS (Fig. 2). Expectedly, for both file systems, a larger size file resulted in enhanced reduction of bacterial biofilm. For example, VB 25/0.06 reduced E. faecalis by 50.8% $\pm$ 3.21 compared to 56.0% $\pm$ 3.71 reduction observed by 30/0.06 (p = 0.0138). Similarly, TS with size 25/0.06 reduced bacterial load by 63.7% $\pm$ 2.53 while size 30/0.06 resulted in 69.5% $\pm$ 2.32 (p = 0.0074) (Fig. 2).

Fig. 2.

Percentage of E. faecalis reduction after instrumentation by either VB or TS file systems. Error bars represent standard deviation. The number 25/0.06 and 30/0.06 represent the file size and taper respectively (p 0.05).

Furthermore, the two file systems were compared. For the same file size and taper, TS showed significantly enhanced reduction in E. faecalis as compared to the VB counterpart (63.7% $\pm$ 2.53 vs 50.8% $\pm$ 3.21 for size 25, and 69.5% $\pm$ 2.32 vs 56.0% $\pm$ 3.71 for size 30) (p 0.0001) (Fig. 3). Overall, TS size 30/0.06 removed about 70% of the inoculated E. faecalis, thereby the most effective file size and system tested in this study (Fig. 3).

Fig. 3.

Percentage of E. faecalis reduction after instrumentation by either 25/0.06 and 30/0.06 sizes of VB or TS file systems. Error bars represent standard deviation (p 0.0001).

In this study, we found that TS file system has removed more E. faecalis from the canal system of the mesial root of the mandibular molar compared to VB file system. E. faecalis was chosen as a model microorganism because of its high prevalence in endodontic infections, and because several previous studies have used the microbial reduction of E. faecalis as a way to analyze different instrumentation techniques and irrigation protocols [4, 12, 20, 21, 22]. E. faecalis plays a major role in endodontic infections due to its ability to form resistant biofilms, invade the dentinal tubules, compete with other microorganisms and low nutrient requirements [8, 9, 23]. Moreover, E. faecalis has a remarkable ability to withstand chemomechanical procedures of root canal cleaning and shaping for both treated and untreated teeth [20, 21]. It can also survive intracanal calcium hydroxide-based medications because of the presence of a proton pump that allows pH homeostasis [23]. Although the maximum microbial reduction was approximately 70%, which differs from some studies that found a reduction of 100% [22, 24, 25], this difference can be explained by difference on methodologies. The studies that achieved complete eradication used culture methods for bacteria detection [22, 24, 25], while our study used qPCR, which is a more sensitive and strategic method to detect and quantify specific targeted bacterial genes [9].

The present ex vivo study was performed to compare the ability of TS and VB file systems to remove E. faecalis from mesial roots and isthmuses of mandibular molars. This type of tooth was selected due to its high complexity, which makes it a common challenge for adequate endodontic cleaning, shaping, and disinfection. Studies have demonstrated that many areas of the root canal remain unistrumented after chemomechanical preparation, regardless of the instrumentation technique used, and these sites can be a safe harbor for bacteria and their toxins [4, 26]. These untouched areas often retain pulp tissue remnants and bacteria [26] that can perpetuate the endodontic disease, thus compromising the success of the treatment. In addition to the anatomical complexity of the mesial canals, the isthmuses may be present in 54–64% of the cases [27, 28]. Isthmus configuration may not be affected by mechanical instrumentation, and disinfection depends mainly on the action of the irrigation solutions. It has been reported that apical diameter of the mesial root canals of mandibular first molar were in the range of 0.24–0.33 mm, so files with apical sizes of 0.25 and 0.30 are the commonly used finishing file sizes [29]. Sodium hypochlorite is the most commonly used root canal irrigant during chemomechanical preparation, and can enter small areas that the instrument cannot reach, thereby improving disinfection [30, 31].

The use of a file with a design that adapts to the anatomical irregularities of the root canal in conjunction with irrigating solutions is an effective method for eliminating, or at least reducing, the microbial load, thus contributing to treatment sucess. The irrigation protocol used in the present study was sodium hypochlorite, due to its potent antimicrobial activity and capacity of dissolution of organic tissues [11, 32]. Qmix® associated with sonic irrigation (EndoActivator) was chosen as the final irrigation solution due to its effective antimicrobial action against E. faecalis [33, 34].

The present study demonstrated that TS produced greater microbial reduction in comparison with VB, independent of the tip size (25 or 30). This finding is in agreement with another study, also using species of E. faecalis, which found that TS was able to remove more bacteria from the root canal when compared to Twisted files [35]. In a recent cone beam computerized tomography (CBCT) study comparing the two systems, it was found that TS can reach a greater area of the canal surface [36]. A viable explanation for this reduction is possibly related the S-shape design of the file, which creates an envelope of motion allowing the file to adapt to the anatomical irregularities of the canal system at smaller apical sizes. TS allows for lower apical transportation and greater dentin conservation than VB [13], and does not induce the formation of new dentin microcracks [37]. Therefore, the TS file design supports the concept of minimally invasive endodontics, which aims to preserve tooth structure and to avoid possible fracture that would result in poor long-term prognosis.

Our study confirmed that the files has succeeded in reducing the bacterial load in the canal system. We have shown for the first time that TS file resulted in a greater reduction than did VB in both file sizes of 25 and 30. While the present study was limited to a single bacterial species, we expect that polymicrobial biofilm to be more resistant to canal instrumentation. To expand on this ex vivo study, future studies with samples collected from patients’ teeth post instrumentation with either of these two file systems could provide stronger evidence on performance of these two files. We hope that our finding would help clinicians make the proper decision in their treatment approach. Further studies on other teeth and root systems such as the mesiobuccal root of maxillary first molar highly recommended.

Within the limitations of this ex vivo study, it can be concluded that TS file system associated with sodium hypochlorite and Qmix® irrigation is more effective than VB in reducing E. faecalis biofilms from mesiobuccal and mesiolingual canals and the isthmuses of mandibular molars. Possibly due to its shaping design, TS allows preparation of canals to smaller sizes, preserving dentin while achieving optimal disinfection.

Conceptualization—RP and SW; methodology—SW, JA and JL; software—MM; validation—MM, SW and RP; formal analysis—SB; investigation—SB, AP, MM and JL; resources—RP; data curation—MM, SW; writing - original draft preparation—MM and SB; writing - review and editing—JK, AP and MM; visualization—SB; supervision—RP and SW; project administration—RP. All authors have read and agreed to the published version of the manuscript.

Not applicable.

Not applicable.

This research received no external funding.

The authors declare no conflict of interest.