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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 2  |  Page : 86-92

Comparative evaluation of efficacy of conventional and passive ultrasonic irrigation with sodium hypochlorite against three endodontic pathogens: An in vitro study


Department of Conservative Dentistry and Endodontics, Bapuji Dental College and Hospital, Davangere, Karnataka, India

Date of Web Publication8-Jan-2018

Correspondence Address:
Dr. J Arun
Room No 6, Department of Conservative Dentistry and Endodontics, Bapuji Dental College and Hospital, Davangere - 577 004, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijohs.ijohs_68_16

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  Abstract 


Introduction: The main goal of endodontic therapy is the complete removal of the pulpal tissues and bacteria within the pulp space to provide an adequate space for three dimensional root canal filling, resulting in a positive environment for the regeneration of periapical tissues. Infections of endodontic origin are treated mainly by means of mechanical procedures aided by chemical substances, of which sodium hypochlorite (NaOCl) is the most commonly used. Different irrigation regimens have been proposed to enhance the effectiveness of NaOCl in disinfecting the root canal system, the use of ultrasonics being one among them. Based on these premises, the purpose of the study is to compare the efficacy of two different irrigation methods in reducing the bacterial count within the root canal.
Objective of the Study: The aim of this study is to compare the efficacy of syringe-needle irrigation to passive ultrasonic agitation in reducing the bacterial count of three endodontic pathogens within the root canal.
Methodology: The study was conducted on 50 extracted human teeth. Teeth were decoronated and the apices were sealed with autopolymerizing resin. The teeth were then inoculated and incubated with a suspension comprising equal amount of three endodontic pathogens: Streptococcus mutans, Candida albicans, and Enterococcus faecalis (E. faecalis). The canal preparations were done using crown down technique and samples were divided into two groups based on irrigation technique: Group 1; Syringe-needle irrigation and Group 2; Additional ultrasonic agitation of irrigant (2.5% NaOCl). The root canals were sampled after the procedure and the samples were inoculated on the respective selective growth media and the colony-forming units (CFUs) were calculated. The statistical analysis used was Student's unpaired t-test for intergroup comparison and one-way ANOVA and Tukey's post hoc test for intragroup comparison.
Results: The Group 2 (ultrasonic agitation) showed highly statistically significant lower CFUs when compared to Group 1 (syringe-needle irrigation). E. faecalis proved to be the most resistant microorganisms of the three used in the study.
Conclusion: Within the limits of the study, ultrasonic agitation of an irrigant proves to be a promising option to achieve significant bacterial reduction and thus achieves reliable and predictable endodontic success.

Keywords: Endodontic pathogens, passive ultrasonic irrigation, sodium hypochlorite, syringe-needle irrigation


How to cite this article:
Arun J, Shenoy A. Comparative evaluation of efficacy of conventional and passive ultrasonic irrigation with sodium hypochlorite against three endodontic pathogens: An in vitro study. Int J Oral Health Sci 2017;7:86-92

How to cite this URL:
Arun J, Shenoy A. Comparative evaluation of efficacy of conventional and passive ultrasonic irrigation with sodium hypochlorite against three endodontic pathogens: An in vitro study. Int J Oral Health Sci [serial online] 2017 [cited 2022 Jan 25];7:86-92. Available from: https://www.ijohsjournal.org/text.asp?2017/7/2/86/222408




  Introduction Top


The root canal treatment of a tooth aims at complete elimination of pathological tissue and microbial pathogens, followed by three-dimensional obturation of the root canal space.[1]

Bacteria and their by-products play an essential role in development and perpetuation of pulpal and periradicular diseases. Primary intraradicular infections are characterized by a mixed consortium dominated by anaerobic bacteria, particularly Gram-negative. Gram-positive anaerobes from genera Peptostreptococcus, Eubacterium, Actinomyces, and facultative or microaerophilic Streptococci are also commonly found. In secondary intraradicular infections, Pseudomonas, Staphylococcus species,  Escherichia More Details coli, Candida species, and Enterococcus faecalis (E. faecalis) are commonly found. E. faecalis is a predominant microorganism found in persistent intraradicular infections.[2] Thus, the irradication of bacteria from the root canal system is important for a successful outcome. Once bacteria are established in the root canal, they cannot easily be reached by the defense mechanisms of the host. Hence, infections of endodontic origin are treated mainly by means of mechanical procedures aided by chemical agents.[3]

By and large, it is impossible to shape and clean the root canal completely using presently available mechanical instruments by themselves. This is mainly due to the intricate and complex anatomy of the root canal system. Irregularities of the root canal wall in particular are a major concern, including oval extensions, isthmi, and apical deltas.[4],[5] These complexities of the root canal system might harbor tissue debris, microbes, and their by-products, which might prevent close adaptation of the obturating material and result in persistent periradicular inflammation. Therefore, irrigation is an essential part of root canal debridement because it allows for cleaning of the root canal system beyond what might be achieved by the root canal instrumentation alone.[6],[7]

Among the various endodontic irrigants known, sodium hypochlorite (NaOCl) is one irrigant that meets most of the requirements of an ideal endodontic irrigant and hence is the most widely used root canal irrigant. NaOCl concentrations ranging 0.5%–5.25% have been used for endodontic irrigation.[8]

For an irrigants effective action, it should be brought into direct contact with the entire canal wall surfaces for a sufficient period. This is true particularly for the apical portions of small root canals. Throughout the history of endodontics, endeavors have continuously been made to develop more effective irrigant delivery and agitation systems for the root canal irrigation.[4],[6] Various irrigant agitation techniques and devices have been introduced which can broadly be classified as manual (syringe irrigation, brushes, and manual dynamic agitation) and machine assisted (sonic, ultrasonic, and pressure alternation devices).[7]

Cleaning and disinfecting intracanal procedures are highly dependent on mechanical and chemical effects of the irrigant. Mechanical effects during irrigation are generated by the flow and backflow of irrigating solution in the root canal. Regardless of the type of irrigant used, the bacterial population inside the root canal is significantly reduced by the mechanical effects of irrigation.[9]

Since the introduction of ultrasonic devices to endodontics by Richman in 1957, they have served as an adjunct to the cleaning and shaping procedures of the root canal system.[6] The ultrasonic files can be activated within the root canal to prepare and debride the root canal mechanically, as well as they can be used passively to enhance the potency of the irrigants.[4] The tissue dissolving and antimicrobial capability of solutions with a good wetting ability are enhanced by ultrasonics when the pulp tissue remnants and/or smear layer are wetted completely by solution and become subjected to ultrasonic irrigation.[4],[5],[6],[7],[8],[9],[10],[11],[12]

Hence, there arises the need for a study that would evaluate the efficacy of conventional irrigation and irrigant activated by passively placing an ultrasonically activated file, against common endodontic pathogens.

Null hypothesis: there is no difference between the antimicrobial efficacy of conventional and passive ultrasonic irrigation (PUI) with 2.5% NaOCl against three endodontic pathogens.


  Methodology Top


Procedure

A total of fifty single-rooted teeth with single canals that were freshly extracted due to periodontal reasons were selected for the study. The teeth were selected irrespective of age and gender of the patient. The selected samples of teeth were caries free and crack free. The teeth were stored in physiological saline until used. The root canals with a curvature >20° were not used for the study. The number of canals and their curvature were determined with the help of radiovisiography images. The rests of periodontal tissue from the external root surface were removed using ultrasonics. The teeth were then decoronated using a diamond disc and the root canals were prepared with K-files, until #20 with 2.5% NaOCl as an irrigant. The teeth were dried with paper points and the apex was sealed with autopolymerizing acrylic resin. Following this, the teeth were sterilized in an autoclave for 15 min at 120°C, and from then on strict asepsis was maintained.

Bacteriology

Three different microorganisms were used in the study: E. faecalis, Candida albicans (C. albicans), and Streptococcus mutans (S. mutans). The test microorganisms were cultured overnight in brain-heart infusion broth. The concentration of the bacterial suspension was approximately set at 105 bacteria/ml. This concentration of bacteria was achieved using 0.5 McFarland scale as standard. Identical volumes of pure cultures of all the three bacteria were then combined to produce a defined mixed culture. Following this, the teeth were inoculated with 10 mL of mixed culture and incubated for 48 h at 37°C.

Enlarging procedures

The root canal length was determined by introducing K file #10 until it slightly surpassed the apical foramen and the working length was corrected by pulling the instrument 0.5 mm back. The root canals were enlarged employing a crown down technique. The coronal and middle third of the root canal were enlarged with Gates Glidden burs #2 and #3 used sequentially shorter, and the apical third of the root canal instrumentation was initiated with K files. Apical preparation was standardized for all teeth to #50. Ethylenediaminetetraacetic acid was used in the canals as the lubricating agent. The canals were irrigated after the use of every instrument with 2 ml of 2.5% NaOCl. Finally, all the canals were rinsed with physiologic saline to eliminate NaOCl from the canals.

Irrigation

The sample teeth were divided into two groups, Group 1 and Group 2 with 25 teeth in each group, based on the irrigation technique employed.

Group 1: Received 2 ml of 2.5% NaOCl irrigation after each instrument used for canal preparation, (total of 10 ml of irrigant per each tooth) with a disposable 5 ml syringe and 26 gauge beveled needle.

Group 2: Received similar irrigation as in Group 1, along with additional agitation of the irrigant with a file #15 fitted on to Suprasson P-max ultrasonic handpiece, placed passively 1 mm short of working length for 10 s, every time the root canal was irrigated.

The final irrigation of all the root canals was done with 2 ml physiological saline.

Sampling procedure

Sterile paper points were selected to sample the bacteria from the root canals. The paper points were selected as per the master apical file size. The bacterial contents of each root canal were sampled with five paper points by placing them in the canal for approximately 1 min and then transferred individually into Eppendorf Tube containing 1 ml of 0.9% physiological saline. The tubes were then shaken in an Eppendorf Mixer for 1 h to detach the bacteria from the paper points. Following this, the suspension was seeded on the selective growth media of the respective three pathogens used. Selective growth media used were Sabaraud dextrose medium, MacConkeys medium, Mitis Salivarius Agar medium for E. faecalis, C. albicans, and S. mutans, respectively.

Statistical analysis

The intergroup comparison was done with Student's unpaired t-test. The intragroup comparison was done using one-way ANOVA test and Tukey's post hoc test.


  Results Top


The results obtained were as follows: The mean and standard deviations (SDs) of colony-forming units (CFUs) for both the groups are mentioned in [Table 1].
Table 1: Mean and standard deviation values of colony forming units for both the groups

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[Table 2] presents the intergroup comparison of mean and SD values of the CFUs for both the groups. The statistical analysis using Student's unpaired t-test for intergroup comparison showed a highly statistical significant difference between the CFUs for all three microorganisms, suggestive of, the ultrasonic agitation (Group 2) to be more efficient in removing the pathogens from the root canal when compared to conventional syringe-needle irrigation (Group 1).
Table 2: Intergroup comparison of mean and standard deviation values

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[Table 3] presents the intragroup comparison of mean and SD values for Group 1. Statistical analysis using one-way ANOVA and Tukey's post hoc test revealed a statistically significant difference between the CFUs of S. mutans and E. faecalis, as well as between C. albicans and E. faecalis, suggestive of E. faecalis being relatively more resistant to shaping and cleaning procedure with conventional syringe needle irrigation. No significant difference was found between the CFUs of S. mutans and C. albicans.
Table 3: Intragroup comparison of mean and standard deviation values for Group 1

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[Table 4] presents the intragroup comparison of Mean and SD values for Group 2. Statistical analysis using one-way ANOVA and Tukey's post hoc test revealed a statistically significant difference between the CFUs of S. mutans and E. faecalis, as well as between C. albicans and E. faecalis, suggestive of E. faecalis being relatively more resistant to shaping and cleaning procedure even after ultrasonic agitation of the irrigant. No significant difference was found between the CFUs of S. mutans and C. albicans.
Table 4: Intragroup comparison of mean and standard deviation values for Group 2

Click here to view



  Discussion Top


Removal of vital and necrotic remnants of pulp tissues, microorganisms, and microbial toxins from the root canal system is essential for endodontic success.[13],[14],[15],[16]

The microorganisms get lodged in areas inaccessible for instrumentation and disinfection, where they grow and multiply. These pathogens are capable of evading host defense mechanisms and cause tissue destruction directly (enzymes, exotoxins, and metabolites) or indirectly (lipopolysaccharide, outer membrane proteins, capsular components, and extracellular vesicles).[2],[16]

S. mutans, C. albicans and E. faecalis, were the endodontic pathogens used in the present study. S. mutans is Gram-positive cocci.[17],[18] This facultative anaerobe is the most frequently isolated microorganism in primary intraradicular infections.[16] It forms a significant part of flora, especially in the coronal part of root canal in carious teeth with exposed pulp chambers.[2]

There is some diversity of species isolated from the root-filled teeth with persistent periapical disease, but there is a consensus among most studies that there is a high prevalence of Enterococci and Streptococci. C. albicans is one among the other species found in higher proportions in individual studies.[19],[20] The prevalence of Enterococci has been a conspicuous finding in all studies that have investigated the flora in the root-filled teeth.[19],[21],[22],[23]

Candida and Enterococci can be viewed as opportunistic pathogens. E. faecalis and C. albicans evade the immune response by a process called sequestration, which involves the formation of a physical barrier between the microbe and the host.[24],[25],[26]

Extracted human teeth were used as representative samples in the study. The teeth were decoronated and the apical third of all the root canals were enlarged to #20 K file, to standardize the length and the apical third width of all the teeth before inoculation. The mechanical preparation of root canal was accomplished using Gates Glidden Drills no. 2 and 3 in the coronal and middle third of the root canal by crown down technique and hand K files for the apical third by a step back technique. Even by means of mechanical instrumentation, only the central body of the canal is shaped leaving the canal fins, isthmi, and cul-de-sacs untouched after the completion of preparation. These areas might harbor tissue debris, microbes, and their by-products, which might prevent close adaptation of obturation material and result in persistent periradicular inflammation. Therefore, irrigation is an essential part of the root canal debridement because it allows for cleaning beyond what might be achieved by the root canal instrumentation alone.[6],[27],[28]

The root canal irrigant used in the study was 2.5% NaOCl. NaOCl has a broad antimicrobial spectrum, dissolves necrotic pulp tissue remnants, inactivates the bacterial toxins, has low surface tension by virtue of which it can reach the areas beyond the reach of instruments and is nontoxic to vital tissues. It is the most commonly used irrigant, as it fulfills most of the requirements of an ideal endodontic irrigant. When in contact with water, NaOCl produces hypochlorous acid and sodium hydroxide. Then, hypochlorous acid produces hydrochloric acid and oxygen. The free chlorine has germicidal properties when it combines with protoplasmic constituents like proteins.[8],[29]

There has been a controversy over the concentration of hypochlorite solutions to be used in endodontics. Concentrations ranging from 0.5% to 5.25% have been advocated for endodontic use. The antibacterial effectiveness and tissue dissolution capacity of aqueous hypochlorite is a function of its concentration, but so is its toxicity.[30] It has been seen in various studies that 5.25% NaOCl causes severe irritation when inadvertently forced into the periapical tissues during irrigation or leaked through the rubber dam. Furthermore, at such high concentrations, it causes erosion of dentin, as well as decreases the elastic modulus and flexural strength of human dentin.[31],[32],[33],[34] Hence, to obtain balanced properties of the irrigant, 2.5% NaOCl was used in the present study.

In the present study, the efficacy of conventional and PUI with 2.5% NaOCl against three endodontic pathogens was evaluated. A statistically significant higher number of CFUs of E. faecalis were seen in Group 1 thus proving to be the most resistant microorganism among the three pathogens tested by means of syringe-needle irrigation. In Group 2, statistically significant higher number of CFUs of E. faecalis were seen thus proving to be the most resistant microorganism among the three pathogens tested in by means of ultrasonic agitation of the irrigant.

When intergroup comparison was done, statistical analysis showed that there existed a highly statistical significant difference between the two groups for all the three pathogens tested with a P < 0.001. The results obtained in the study suggest that the ultrasonic agitation of the irrigant results in highly significant reduction in microbial count within the root canal when compared with that of conventional syringe-needle irrigation. It was also seen that E. faecalis was the most resistant microorganism and showed significantly higher CFUs when compared to C. albicans and S. mutans.

The results of the study are consistent with the studies done by Huque et al.[35] 1998, Spoleti et al.[10] 2003, and Weber et al.[36] 2003, wherein it was seen that PUI resulted in significant bacterial reduction when compared to syringe needle irrigation.

Conventional irrigation with syringes has been advocated as an efficient method of irrigant delivery. Nevertheless, the mechanical flushing action created by conventional handheld syringe-needle irrigation is relatively weak. A previous study has shown that when conventional syringe-needle irrigation was used, the irrigating solution was delivered only 1 mm deeper than the tip of the needle.[37] This is a disturbing issue because the needle tip is often located in the coronal third of a narrow canal or at best, the middle third of a wide canal. The penetrating depth of irrigating solution and its ability to disinfect dentinal tubules are therefore limited. Hence, this could be the reason why the microbial reduction in the syringe-needle irrigation group was highly significantly lesser than the ultrasonic irrigation group in the present study.[37]

In the Group 2, the irrigant was agitated with a #15 K ultrasonic file that was placed passively in the root canal, 1 mm short of the working length, and activated at a frequency set at 25–30 kHz for 10 s. The term “PUI” is used because the file is held in the canal in such a way that it does not touch the walls of the root canal and thus does not result in active cutting of root dentin. The highly statistical significant reduction of microorganisms in the ultrasonic agitation group when compared to the syringe-needle irrigation group could be due to the following reasons:

  1. The file operates in a transverse motion, setting up a characteristic pattern of nodes and anti-nodes along its length. This produces acoustic streaming and cavitation of the irrigant. This in-turn increases the velocity and volume of the irrigant flow within the root canal, thus making it possible for the irrigant to reach areas of canal irregularities [38]
  2. High-power ultrasound causes deagglomeration of bacterial biofilms through the action of acoustic streaming. Deagglomeration of biofilms within the root canal might render the resulting planktonic bacteria more susceptible to bactericidal activity of NaOCl [4]
  3. Cavitation may have produced temporary weakening of the cell membrane, making the bacteria more permeable to NaOCl [6]
  4. Ultrasonic waves accelerate chemical reactions and potentiate the bactericidal efficacy of NaOCl.[39]


In the present study, E. faecalis was the most resistant microorganism of the three pathogens tested in both the groups. The capability to endure prolonged periods of nutritional deprivation, capability to alter the host response, suppress the action of lymphocytes, the utilization of serum as a nutritional source, and ability to resist the intracanal medicaments are the main survival and virulence factors of E. faecalis.[25] These survival and virulence factors explain why in both the study groups, E. faecalis was relatively more resistant to cleaning and shaping procedures.

In studies, comparing depth of irrigant penetration into root canals and the ability to produce clean and debris-free root canals using various irrigant agitation techniques, PUI has proved to be one of the most effective means of achieving the goal.[40],[41]

In an in vitro study done by Bago Jurič et al., laser-activated irrigation, PUI, and RinseEndo were equally effective in eliminating E faecalis from root canals, inferring that PUI is at par with the contemporary means of irrigation techniques for eliminating microflora from root canals.[42]

In an in vitro study done by Bhardwaj et al., results showed that 1% NaOCI with PUI was effective in completely removing E. faecalis biofilms from root canal walls, proving the potential of PUI as one of the most effective means of irrigant agitation techniques.[43]

Further in vivo studies are required in this field to establish more and higher level of scientific evidence regarding the use of PUI for better antimicrobial activity of the irrigant and for a better successful outcome of endodontic therapy.


  Conclusion Top


The proposed null hypothesis was rejected.

Within the limitations of the present study, it can be concluded that:

  1. PUI of the root canal system has shown very promising results to increase the antimicrobial efficacy of a root canal irrigant irrespective of the infective microorganism and hence can be used as an adjunct in root canal treatment to achieve more predictable and reliable endodontic outcome
  2. Among the three endodontic pathogens tested in the study, E. faecalis was the most resistant microorganism to available cleaning and shaping procedures.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflflicts of interest.



 
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Bhardwaj A, Velmurugan N, Sumitha. Ballal S. Efficacy of passive ultrasonic irrigation with natural irrigants (Morinda citrifolia juice, Aloe Vera and Propolis) in comparison with 1% sodium hypochlorite for removal of E. faecalis biofilm: An in vitro study. Indian J Dent Res 2013;24:35-41.  Back to cited text no. 43
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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