Evaluation of Microbial Reduction in Root Canals Instrumented with Reciprocating and Rotary Systems

Abstract

Objective

This in vitro study aimed to evaluate the efficacy of the disinfection of root canal systems carried out with Reciproc^TM and ProTaper Universal^TM systems using 1% sodium hypochlorite (NaOCl).

Methods

Forty human single-rooted mandibular premolars were infected with Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, and twenty were not infected. The specimens were randomly divided into 6 groups (n = 10): Group 1: ProTaper Universal^TM + 1% NaOCl; Group 2 (positive control): ProTaper Universal^TM + saline; Group 3 (negative control without microorganisms): ProTaper Universal^TM + saline; Group 4: Reciproc^TM + 1% NaOCl; Group 5 (positive control): Reciproc^TM + saline; Group 6 (negative control without microorganisms): Reciproc^TM + saline. Samples were collected before and after the completion of specific treatments, and plated in specific media cultures. The Fisher exact test was used for the statistical analysis of differences in terms of presence or absence of microbial growth among groups. For all tested pathogens, significant differences (p < 0.001) were verified between the instrumentation systems used.

Results

ProTaper Universal^TM associated with 1% NaOCl completely eliminated all microorganisms. Microbial growth, however, was observed when Reciproc^TM was used associated with 1% NaOCl.

Conclusion

According to the protocol executed for this study, the Reciproc^TM system associated with 1% NaOCl was not able to completely eliminate E. faecalis, P. aeruginosa, S. aureus and C. albicans from the root canal systems.

Introduction

One of the most important goals of endodontic treatment is to significantly eliminate or reduce the microbial load present inside an infected root canal system (1). However, the total elimination of microorganisms in the root canal remains a difficult task. Pathogens such as Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans are frequently observed when endodontic treatment has failed (2).

Several methods, including the use of a variety of instrumentation techniques (3), irrigation schemes with antimicrobial solutions (4) and intracanal medications (5), have been described aiming at a more effective intracanal disinfection.

Sodium hypochlorite (NaOCl) is the most common irrigating solution used during chemomechanical preparation due to its broad spectrum of antimicrobial activity and its ability to dissolve organic material. The efficiency of disinfection by NaOCl is related to its contact time with the microorganisms and its concentration, which can range from 0.5% to 5.25% (6).

In recent years, several nickel-titanium (NiTi) instruments have been designed for root canal preparation with continuous rotation movement. ProTaper Universal^TM system (Dentsply Maillefer, Ballaigues, Switzerland) is one of the most popular endodontic NiTi systems currently on the market. Its technique is based on the sequential use of files to clean and shape the root canal adequately (7, 8).

Recently, a new single-file instrumentation system with reciprocating motion has been introduced. The Reciproc^TM system (VDW, Munich, Germany) utilizes a single instrument to prepare the root canal, thus reducing the work time, making it four times faster than traditional NiTi systems, providing greater comfort for patient and professional (9).

A concern has been presented, however, regarding the ability of a single-file instrumentation system to disinfect the root canal, due to the shortening of preparation time of the canal, together with the lesser amount of antimicrobial agent and shorter contact time (10, 11).

Thus, although several previous studies have evaluated the cleaning efficacy (12), the apical extrusion of debris (13), and the reduction of endotoxins (14) of single-file systems, only few studies (10, 11, 15, 16) have analyzed the ability to reduce intracanal microbial agents when associated with antimicrobial irrigating solutions.

Therefore, the aim of this in vitro study was to evaluate the efficacy of the Reciproc^TM and ProTaper Universal^TM instrumentation systems to disinfect root canals infected with E. faecalis, P. aeruginoas, S. aureus e C. albicans using 1% sodium hypochlorite.

Materials and Methods

Sample selection and preparation

Sixty freshly intact human mandibular premolar teeth (length 20-21 mm), straight, with radiographically confirmed single root canal and fully formed apices, were obtained from the Human Tooth Bank of the Prosthodontics and Oral and Maxillofacial Surgery, Dental School, Federal University of Pernambuco, Brazil, for this study after approval by the Research Ethics Committee of the University’s Center of Health Sciences. The teeth were stored in 10% formalin (INDALABOR, Minas Gerais, Brazil) until use.

The coronal access was performed. To determine the working length (WL), a #10 K-file (Dentsply-Maillefer, Ballaigues, Switzerland) was inserted into the root canal until it was visible at the apical foramen. The WL was calculated to be 1 mm less than the length obtained with this initial file.

The specimens were stored in glass test tubes and were individually sterilized in an autoclave at 121 °C for 30 min. Ten samples were randomly chosen and immersed totally in bottles containing 10 mL of autoclaved Brain Heart Infusion (BHI, Oxoid, Basingstoke, UK). They were kept in an incubator at 37 ± 1 °C for 96 h to check the sterilization’s efficacy.

Experiment preparation

The methodology used has been described previously by Câmara et al. (17). Glass vials with rubber stoppers were adjusted for use in the present experiment. The experimental systems were sterilized in an autoclave at 121 °C for 30 min, and inside a laminar flux chamber (Veco, Piracicaba, Brazil), they were filled with BHI (Oxoid, Basingstoke, UK). Then the experimental systems were kept in an incubator at 37 ± 1 şC for 96 h and no turbidity of the medium was observed.

Microbial strains

The microorganism strains used in this experiment were obtained from the American Type Culture Collection™ (ATCC, Rockville, MD, USA): Enterococcus faecalis (ATCC 6057), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213) and Candida albicans (ATCC 10231).

Microbial cultures and root canal infection

The following procedures were performed inside a laminar flux chamber (Veco, Piracicaba, Brazil) using sterilized instruments and materials. Isolated 24 h colonies of pure cultures of E. faecalis, P. aeruginosa, S. aureus and C. albicans grown on 10% sheep blood plus BHI (Oxoid, Basingstoke, UK) agar plates were suspended in a sterile 0.85% NaCl solution. The suspensions of the bacteria had their optical density adjusted spectrophotometrically to approximately 3.0 x 10⁸ colony-forming units (CFU) mL^-1, and the suspensions of C. albicans had their optical density adjusted spectrophotometrically (FEMTO, Săo Paulo, Brazil) to a higher concentration of 3.0 x 10⁹ CFU mL^-1. From each experimental suspension, 1 mL was removed and a mixture of the four selected microorganisms was prepared.

The sterilized experimental systems were then opened. The root canals were infected, except for the negative control, with 10 μL of the suspension containing the microorganisms using an automatic micropipette (Gilson, Villiera-le-Bel, France) placed into the access cavity of each tooth. After introduction of the suspension, sterile #10 K-files (Dentsply-Maillefer, Ballaigues, Switzerland) were used to carry the microbial suspension to the WL. The infected teeth were incubated at 37 ± 1 şC for 48 h. The turbidity of the medium during the incubation period indicated microbial growth. The purity and the identification of the cultures were confirmed by Gram staining, colony morphology and the growth on Petri dishes with the media: Bile Aesuclin Azide Agar (Merck KGaA, Darmstadt, Germany) to verify the presence of E. faecalis, Cetrimide Agar (Merck KGaA, Darmstadt, Germany) to verify the presence of P. aeruginosa, Mannitol Salt Phenol-red Agar (Merck KGaA, Darmstadt, Germany) to verify the presence of S. aureus, and Saboraud Dextrose Agar (Merck KGaA, Darmstadt, Germany) to verify the presence of C. albicans. If the 4 microorganisms were not identified, the experimental system was discarded. The efficiency of the method for the infection of the root canal was observed in a pilot study. All assays were conducted in triplicate under aseptic conditions.

Experimental groups

The infected teeth were removed from the experimental systems with the contaminated medium and transferred to glass vials without the medium, in order for the teeth to remain fixed at the beginning of the instrumentation.

The specimens were randomly divided into 2 experimental groups (groups 1 and 4) and 4 control groups (groups 2, 3, 5 and 6) with 10 root canals each according to the protocol used during root canal preparation, as follows:

• Group 1: 1% NaOCl + ProTaper Universal^TM system;

• Group 2 (positive control): sterile 0.85% NaCl solution + ProTaper Universal^TM system;

• Group 3 (negative control, without microorganisms): sterile 0.85% NaCl solution + ProTaper Universal^TM system;

• Group 4: 1% NaOCl + Reciproc^TM system;

• Group 5 (positive control): sterile 0.85% NaCl solution + Reciproc^TM system;

• Group 6 (negative control, without microorganisms): sterile 0.85% NaCl solution + Reciproc^TM system.

Irrigation

For irrigation of the root canal, the 3-mL FCF syringe system (FCF, Săo Paulo, SP, Brazil) with a 30-gauge needle (Injecta, Diadema, SP, Brazil) was used.

Freshly prepared 1% NaOCl (Farmácia Escola Carlos Dumont de Andrade, Recife, PE, Brazil), and sterile 0.85% NaCl solution were used for irrigation in biomechanical preparation of the root canals, with the 30-gauge needle demarcated at 1 mm short of the apical foramen.

Root canal biomechanical preparation

The ProTaper Universal^TM instruments were operated with an electric motor (Driller Endo-Pro Torque, Sao Paulo, Brazil) at a speed of 300 rpm as follows: SX file was used to one half of the WL; S1 file was used up to 4 mm short of the apex; S1 and S2 files were used to the full WL; and F1 (20.07), F2 (25.08) and F3 (30.09) files were used to the full WL. For irrigation, 5 mL of irrigating solution was used before instrumentation and after each instrument use, and so, the total volume of irrigant was 35 mL per canal.

The Reciproc^TM instruments were used with a contra angle low speed (Sirona Dental Systems GmbH, Bensheim, Germany) coupled to the electric motor for reciprocating kinematics (VDW Silver; VDW GmbH, Munich, Germany). Each root canal was instrumented with a single VDW Reciproc^TM (VDW, Munique, Alemanha) R40, having a size 40 at the tip and a taper of 0.06 over the first 3mm. Initially the canal was irrigated, and then, the Reciproc^TM instrument was introduced with a slow in-and-out pecking motion of about 3 mm in amplitude with a light apical pressure. After three pecking motions, the instrument was removed from the canal, cleaned and the canal was irrigated. These procedures were repeated until the WL was reached by the instrument. After instrumentation was completed, the canals were irrigated once again. Each instrument was used to prepare only one canal. The total volume of irrigating solution was 10 mL per canal.

A single operator instrumented all root canals. After the instrumentation, irrigation with 5% sodium thiosulphate solution (Synth^®, Săo Paulo, Brazil) was used to neutralize NaOCl. The shaping time of the root canals was recorded.

Assessment of antimicrobial action of the irrigants

Samples from each root canal were tested to verify the presence or absence of the microbial growth both before and after final disinfection procedures. To assess the antimicrobial action of the instrumentation protocols, sterile paper points size 15 (Dentsply-Maillefer, Petrópolis, Brazil) were consecutively placed in the root canal. Each paper point was left in the root canal for 1 min, as follows: X1 (before biomechanical preparation) and X2 (after final disinfection). The paper points were transferred to Petri dishes containing the following media: Bile Aesuclin Azide Agar, Cetrimide Agar, Mannitol Salt Phenol-red Agar, and Saboraud Dextrose Agar.

The plates were then incubated at 37 ± 1 şC for 48 h. After incubation, microbial growth was assessed with light microscopy (Leica Microsystems, Săo Paulo, Brazil) at 400×.

Statistical analysis

The categorical data were summarized by means of absolute frequency and relative percentage. The results were analyzed statistically using Fisher’s exact test. A level of significance of .05 was adopted. The Statistical Package for the Social Sciences, version 21 (SPSS, Chicago, IL) was used.

Results

Irrigant antimicrobial efficacy

Microbial growth was observed in all initial samples, except for the negative control groups, demonstrating that the contamination was effective in all root canals for all experimental groups.

All positive control samples showed microbial growth before and after biomechanical preparation, whereas the negative control samples showed no microbial growth.

The antimicrobial efficacy of treatments in each group against E. faecalis, P. aeruginosa, S. aureus and C. albicans is shown in Table 1.

TABLE 1. Antimicrobial efficacy of various treatment protocols in root canals infected with E. faecalis, P. aeruginosa, S. aureus e C. albicans.

Group	Presence		Absence		TOTAL		P value
	n	%	n	%	n	%
1% NaOCl + ProTaper UniversalTM	-	-	10	100.0	10	100.0	p (1) < 0.001*
Saline + ProTaper UniversalTM (positive control)	10	100.0	-	-	10	100.0
Saline + ProTaper UniversalTM (negative control)	-	-	10	100.0	10	100.0
1% NaOCl + ReciprocTM	10	100.0	-	-	10	100.0
Saline + ReciprocTM (positive control)	10	100.0	-	-	10	100.0
Saline + ReciprocTM (negative control)	-	-	10	100.0	10	100.0
TOTAL	30	50.0	30	50.0	60	100.0

* Statistically significant difference at .05.

The analysis of the frequency of presence or absence of microbial growth according to the treatment applied revealed the existence of statistically significant differences (p < 0.001) between the instrumentation systems used.

In the group where the canals were instrumented using the ProTaper Universal^TM system associated with irrigation with 1% NaOCl, the complete elimination of all microorganisms was verified. However, in the group where the Reciproc^TM system associated with 1% NaOCl was used, microbial growth was observed for all microorganisms tested.

Figure 1 shows the antimicrobial activity presented by ProTaper Universal^TM and Reciproc^TM systems associated with 1% NaOCl against (A) E. faecalis, (B) P. aeruginosa, (C) S. aureus, e (D) C. albicans, respectively.

FIGURE 1.

Antimicrobial activity presented by ProTaper Universal^TM and Reciproc^TM systems associated with 1% NaOCl against (A) E. faecalis, (B) P. aeruginosa, (C) S. aureus, e (D) C. albicans, respectively.

Root canal preparation times

The mean preparation times were 4.2 ± 1.5 minutes for ProTaper Universal^TM system, and 40 ± 80 seconds for Reciproc^TM system.

Discussion

The purpose of this study was to evaluate the efficacy of the Reciproc^TM and ProTaper Universal^TM instrumentation systems together with 1% sodium hypochlorite to disinfect root canals experimentally contaminated with various pathogens.

E. faecalis, P. aeruginosa, S. aureus e C. albicans were selected as the microbiological markers because they are considered the most resistant species in infected root canals, and are often associated with endodontic treatment failures (2).

Among the irrigating solutions currently used, NaOCl is the most common and accepted worldwide due to its properties that contribute to an effective chemomechanical preparation. As demonstrated in this research as well as in studies of Gulsahi et al. (4), Almeida et al. (18) and Shantiaee et al. (19), NaOCl has a broad spectrum of antimicrobial activity, has the capacity to dissolve organic material, and acts as a lubricant during root canal system instrumentation. However, there is no general agreement regarding its optimal concentration, contact time and temperature for clinical use in Endodontics.

The antimicrobial action of NaOCl increases with its concentration, ranging from 0.5% to 5.25%, but this is also accompanied by an increase in its toxicity (20). The NaOCl concentration used in this study was 1%, since previous research (17, 21) indicates this as the optimal concentration for clinical use. Higher concentrations, according to this research, do not have better bactericidal capacity and lead to a higher degree of aggression to the periapical tissues.

Several studies (4) have evaluated the contact time required by NaOCl to eliminate different microorganisms. Vianna et al. (22) demonstrated that the time required by 1% NaOCl to eliminate suspensions of E. faecalis, S. aureus, and C. albicans was 20 minutes. Whereas Radcliffe et al. (23) verified that the same concentration of irrigant required 10 minutes to promote negative cultures of suspensions of E. faecalis. However, Retamozo et al. (24) observed that 1.3% NaOCl was not able to disinfect dentin cylinders contaminated with E. faecalis even after 40 minutes of irrigation. On the other hand, Câmara et al. (17) demonstrated that even after 4.2 ± 1.5 minutes of preparation time, 1% NaOCl was effective in disinfecting root canals infected with E. faecalis, P. aeruginosa, S. aureus e C. albicans. These findings corroborate the results of the present study, where the efficiency of this solution in the group in which the ProTaper Universal^TM system has been used was observed.

Single-file instrumentation techniques have been proposed for the preparation of root canal systems. The concept of using a single instrument to prepare the root canal is interesting as it can considerably reduce the working time, providing greater comfort for patient and professional. However, a concern with these systems refers to their ability to disinfect the root canal, since, due to the shortening of the preparation time of the canal, minor amounts of irrigating solution, less permanence time of the solution in the canal, or all of these may hamper the eradication of all microorganisms from the root canal system (10, 11).

The results of this study demonstrated that the chemomechanical preparation using Reciproc^TM system associated with 1% NaOCl was not effective in promoting samples with absence of microbial growth of the various pathogens studied. Nevertheless, the ProTaper Universal^TM system was effective in eliminating all microorganisms from root canals when used the same concentration of irrigating solution.

The results of this research did not agree with the findings obtained by previous studies such as those by Alves et al. (10), Basmaci, Oztan e Kiyan (11), Martinho et al. (14), Machado et al. (15) and Siquera et al. (16). Basmaci, Oztan and Kiyan (11) verified no significant differences in microbial reduction between single-file techniques (Self-Adjusting File^TM e Reciproc^TM) and conventional Ni-Ti instrumentation (ProTaper^TM) when irrigation with 5% NaOCl combined with 15% EDTA or 7% maleic acid was employed. Siqueira et al. (16) also verified similar disinfecting capabilities of the single-file instrumentation systems (Self-Adjusting File^TM and Reciproc^TM) and rotary (Twisted File^TM) when combined with 2.5% NaOCl irrigation.

It is important to emphasize, however, that, differently from the studies cited, in our research both instrumentation systems used the same concentration of irrigating solution, but the average root canal preparation time and the volume of irrigating solution were different, varying according to the instrumentation system employed. In the groups instrumented with the Reciproc^TM system, a total of 10 mL of NaOCl with a mean instrumentation time ranging from 40 ± 80 seconds was employed. Whereas, when the ProTaper Universal^TM system was used, a mean time of 4.2 ± 1.5 minutes was used to prepare the canal with 35 mL of NaOCl.

Another important point that can be emphasized is regarding the differences in taper and tip sizes existent among tested instrumentation systems. There are controversies about whether, in fact, the diameter of the apical preparation can significantly influence the outcomes of root canal disinfection (14), since smaller diameters provide a greater amount of untouched areas of the canal (25). In this study, despite the larger tip diameter presented by the Reciproc^TM R40 (40.06) instrument compared to the ProTaper Universal^TM F3 (30.09), allowing a slightly larger apical preparation, was not able to provide a superior intracanal disinfection when associated with 1% NaOCl.

When comparing a single-file instrumentation system (Reciproc^TM) with a NiTi mechanical-rotary system (BioRaCe^TM), Alves et al. (10) reported that the Reciproc^TM system may be equivalent to other NiTi systems since the width of the apical preparation, the volume of irrigating solution and the duration of irrigation are also similar.

With respect to this, the present study found that the mean time of root canal preparation and the volume of irrigating solution used play a fundamental role in the antimicrobial effectiveness achieved during chemomechanical preparation using different instrumentation systems. Thus, the results presented here emphasize the importance of developing appropriate protocols to be obeyed during the use of single-file systems aiming a more effective clinical practice.

Conclusion

In conclusion, this study showed that the two instrumentation techniques presented significant differences in the ability to disinfect root canals. According to the protocol used, the single-file instrumentation system (Reciproc^TM) in combination with 1% NaOCl was not able to eliminate completely E. faecalis, P. aeruginosa, S. aureus e C. albicans.