INTRODUCTION:

Microorganisms are an important cause of failure in Endodontic treatment.¹ The positive result of the Endodontic treatment relies on the reduction or absence of microorganisms present in the root canal.² Dentin pulpal tissue, bacteria, and tooth debris may cause surgical or nonsurgical irregularities in root canal systems, even after careful mechanical preparation.³Therefore, irrigant solutions are used in combination with Endodontic therapy.^4-7Chemomechanical preparation, including both mechanical and chemical irrigation, is significant for eliminating bacterial populations.⁸

Mechanical instrumentation alone is insufficient to yield effective disinfection ⁹because the complexity of root canal anatomy^10,11, prevents the accessibility of instrumentation and provides a shelter for microorganisms¹². Sodium hypochlorite (NaOCl) is the most frequently used irrigant solution during treatment because of its antimicrobial property^13,14 and tissue-dissolving¹⁵. Several concentrations of NaOCl ranging from 0.5%–5.25% were found in the endodontic literature, and the most widely used concentration is 2.5 ¹⁶. Strong antimicrobial activity¹⁷, tissue-dissolving properties by high concentration Sodium hypochlorite. It also lead to increased cytotoxicity¹⁸ and periapical tissue irritation¹⁹.

Chlorhexidine (CHX) is a substitute irrigant to NaOCl because of its broad-spectrum antimicrobial activity and considerably less toxic than NaOCl²⁰. The most widely used concentration of CHX for root canal therapy is 2%²¹. In contrast to Sodium hypochlorite, high concentrations of CHX provide bactericidal effect, whereas low concentrations provide only a bacteriostatic effect²². CHX can be used either as a gel or solution with the same effectiveness. The mechanism is due to its substantivity; the positive charges of the CHX molecule bind to the negative charges in dental surfaces by lengthened adherence, which in turn leads to long-lasting antimicrobial activity. However, as an endodontic irrigant, the lack of tissue-dissolving capacity of CHX is a considerable drawback²³. MTAD is a combination of materials developed for use in endodontic treatment as a final irrigant²⁴. It is a mixture of Doxycycline [a tetracycline isomer], citric acid, and Polysorbate80 [a detergent]²⁵. The efficacy of MTAD as a conditioner in periodontal treatment is still under investigation.²⁶

PAD [Photoactivated disinfection] is a newer antimicrobial strategy²⁷ involving a combination of non-toxic Photosensitisers²⁸ or dyes and non-harmful visible light sources to disinfect the root canal.^29,30 Power laser in particular not bactericidal but useful for photochemical activation of oxygen-releasing dyes leading to cell membrane and DNA damage to microorganisms³¹. PAD technique can be undertaken with a range of visible red and near-infrared lasers and dyes such as toluidine blue, methylene blue, chlorine p6, etc.Candida albicans and Enterococcus faecalis most common species isolated from the root canals with secondary apical periodontitis. Many in-vitro studies related to the Antimicrobial activity of root canal irrigants against microorganisms are in the literature^32-34. In the current study, the efficacy of five groups of irrigantsis tested against three major pathogens, a combination of adjuvant and an irrigant used which was not found in the literature. Therefore, this study evaluates the effectiveness of NaOCl 0.5%, PAD alone, and PAD with chlorhexidineand MTAD.

MATERIALS AND METHODS:

The following MTCC strains of Enterococcus faecalis, Staphylococcus aureus, and Candida albicans were grown on BHI broth and checked for turbidity with 0.5 Macfarlandstandard. Agar Disk diffusion was performed on Muller Hinton Agar plate, 6mm well cut, and the 100ug concentration of the medicaments was added and kept for diffusion for 2hrs and the organisms were lawned over the plate incubated for 24hs at 37⁰C and checked for Zone of Diameter of Inhibition.

Root canal Irrigants:

The irrigants used were NaOCl 3%, 2% Chlorhexidine, MTAD solution mixture of a solution of Tetracycline, 4.25% citric acid, and 0.5% polysorbate (Tween 80) Hi-media. 100µm of PS solution of chlorine p6 added to the inoculums kept for 20mins and 2% CHX 5ml added³³.

Statistical Analysis:

To identify the significant group the data was analyzed by One way-ANOVA and Bonferroni Posthoc Test (p<0.05)

Results:

The data summarized in Table 1 show that the highest zone of inhibition by MTAD and PAD along with chlorhexidine and for all three organisms compared with Positive control, followed by chlorhexidine, 3% NaOCl. PAD had less inhibition zone showing its less Antimicrobial properties, but when added with Chlorhexidine had high effects.C. albicans was more inhibited by PAD +CHX with a 22mm Zone of inhibition followed by MTAD with a 20mm Zone of inhibition, and less inhibition by PAD with a 0.7mm Zone of inhibition. Enterococcus faecalis was more inhibited by MTAD with a 35mm Zone of inhibition followed by PAD +CHX with a 19 mm Zone of inhibition, and less inhibition by PAD with a 12mm Zone of inhibition. Staphylococcus aureus was more inhibited by MTAD with a 25mm Zone of inhibition followed by PAD+CHX with a 20mm Zone of inhibition, and less inhibition by PAD with a 15mm Zone of inhibition.From the evaluated study MTAD and PAD with Chlorhexidine had more effect on the assays of organism. MTAD is more effective in S.aureus and E.faecalis and less in C.albicans whereas PAD+CHX was more effective in Candida and less in S.aureus and E.faecalis. MTAD and PAD with CHX statistically showed a significant difference in inhibitory activity with p<0.05 among different organisms, whereas PAD and CHX were not statistically significant. (Table 1; Figure 2,3.4)

Fig. A Fig. B

Fig. C

Figure 1: MTCC strains of E.faecalis (A), S. aureus (B), and C.albicans (C)

Fig. D

Fig. E

Figure 2: Zone of Inhibition of S.aureus against Irrigants

(Fig D&E)

Figure 3: Zone of inhibition of Candida albicans against Irrigants

Figure 4: Zone of Inhibition of E.faecalis.

DISCUSSION:

Irrigation plays an important role in successful debridement and disinfection. In this study, all the tested irrigant solutions exhibit an inhibitory effect on the tested strains. For a successful Endodontic treatment, the effect of the irrigant plays a major role. The NaOCl solution is a highly recommended irrigant, but the optimal concentrations still a quire³⁵ toxicity and irritation in periapical and periodontal tissues seen in higher concentrations³⁶. 0.3% NaOCl is an acceptable cytotoxic level but this concentration is less effective. The CHX has been a representative irrigant in both periodontal and endodontic therapy for many years due to low cytotoxicity³⁷ and antimicrobial properties³⁸. MTAD has been described as a worldwide irrigating solution , it was first used as an alternative to EDTA to remove the smear layer. MTAD has been used in dentistry as a final irrigant for the removal of the smear layer. Many studies have proven that MTAD is effective in killing resistant micro-organism and assists in antimicrobial activity³⁹. Among all tested irrigants MTAD, showed the highest zone of bacterial inhibition maximum of 35mm to E.faecalis⁴⁰. the present study correlates with various studies where MTAD had better action than 3% sodium hypochlorite⁴¹. Under PDA sensitizer dye solution the zone of inhibition (14±0.9mm) which was similar to the study where unable to kill alone. In the present study PAD combined with CHX showed high inhibition to all tested strains and without any investigation can be used in pre-root canal treatment which was similar in many related studies⁴². CHX had shown moderate inhibition to the organisms but when compared to MTAD, PAD+CHX similar to the study MTAD and chitosan had more inhibitory effect than CHX⁴³. Soukos et al showed PAD to be 97% effective in reducing E.faecalis when applied alone, but in our study, it was the second least with Candida being less inhibitory.⁴⁴

The most common pathogen within the root canal of teeth with persistent periapical lesions is C.albicans and it is Symbiosis with another major pathogen E.faecalis⁴⁵ there is abundant evidence that getting a sterile toothafter treatment is impossible thus intracanal medical irrigants after chemomechanical preparation in disinfection of root canal system is important⁴⁶. Of the tested organisms Candida was more sensitive to all the irrigants followed by S.aureus and E.faecalis in in the study. Candida and E.faecalis were effectively killed by CHX with a zone of 19mm than S.aureus with a zone of 15mm. Torabinejad et al showed that MTAD is significantly more effective than CHX and NaOCl in killing Candida and E.faecalis, similar to our study MTAD had a zone of inhibition of 35mm.⁴⁷

LIMITATIONS OF THE STUDY:

The Limitation of the study includes the death of microorganisms using irrigants cannot be evaluated in an agar diffusion test. As the properties of irrigants may vary inside the root canal due to Dentin materials. So in-vitro Antibacterial results have to be validated by In-vivo tests to prove its efficacy.

CONCLUSION:

Within the limitation of this in-vitro study, it is concluded that MTAD and PAD along with CHX showed the highest Antibacterial and Antifungal activity among all irrigants. Thus PAD used along with CET has inhibitory activity on pathogens hence it can be recommended as an adjuvant for thorough disinfection and sterilization of the dental root canal system.

CREDIT AUTHORSHIP CONTRIBUTION STATEMENT:

Authors 1, 2: Methodology, Investigation, Funding acquisition. Author 2: Methodology, Investigation ,Formal analysis. Author 3: Formal analysis, Supervision. Author 4: Formal analysis: Formal analysis, Supervision. etc.

DECLARATION OF COMPETING INTEREST:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Received on 04.10.2023 Modified on 20.02.2024

Research J. Pharm. and Tech 2024; 17(8):3752-3756.

DOI: 10.52711/0974-360X.2024.00583

Organism	PAD	PAD+CHX	CHX	MTAD	Positive control	Negative control
C.albicans	0.7mm	22mm	19mm	20mm	21mm	-
S.aureus	15mm	20mm	15mm	25mm	19mm	-
E.faecalis	12mm	19mm	18mm	35mm	17mm	-