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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 9  |  Issue : 3  |  Page : 63-71

Evaluation of antimicrobial action, pulp tissue dissolution, and smear layer removal of an experimental irrigant combining Salvadora persica and sodium hypochlorite with and without ultrasonic activation: An in vitro study


1 Post Graduate Student, Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India
2 Reader, Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India
3 Professor, Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India
4 Professor and Head of Department, Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India

Date of Submission29-May-2022
Date of Acceptance06-Jul-2022
Date of Web Publication28-Sep-2022

Correspondence Address:
Dr. V A Jibin Karim
Venat House, Eriyad P.O., Kodungallur, Thrissur, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpcdr.ijpcdr_16_22

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  Abstract 


Aim: To evaluate the pulp tissue dissolution, antimicrobial action, and smear layer removal using an experimental irrigant combining Salvadora persica and sodium hypochlorite (NaOCl) with and without ultrasonic activation in comparison to standard irrigation protocol of NaOCl and ethylenediamine tetraacetic acid (EDTA) with and without ultrasonic activation.
Materials and Methods: Pulp tissue dissolution - Pulp tissue weighing 0.9 g was dissolved in each experimental Group G1: 5 ml of 5% NaOCl, G2: G1 + ultrasonic activation, G3: 5 ml 5% NaOCl + 40% S. persica, G4: G3 + ultrasonic activation and weighed after 1, 5, and 10 min. The difference in weight of the tissue sample was assessed. Smear layer removal and calcium/phosphate (Ca/PO4) ratio - G1: 5 ml of 5% NaOCl, saline, 1 ml of 17% EDTA, G2: G1 + ultrasonic activation, saline, 17% EDTA, G3: 5 ml 5% NaOCl + 40% S. persica G4: G3 + ultrasonic activation. Each tooth was then split longitudinally and subjected to scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM-EDX) analysis. Antibacterial Activity- assessed against Enterococcus Faecalis.
Statistical Analysis: SEM, pulp tissue dissolution and antimicrobial efficacy – Kruskal–Wallis and post hoc Mann–Whitney EDX – ANOVA and Tukey's honestly significant difference post hoc analysis.
Results: Pulp dissolution by G3 and G4 was better. However, there was no statistically significant difference between the groups (P > 0.05). Experimental combination performed better in terms of smear layer removal, showed higher amount Ca/PO4 in EDX analysis, but the difference was not statistically significant between the groups (P > 0.05). G2 showed maximum antimicrobial efficacy compared to all groups but not statistically significant (P > 0.05).
Conclusions: The combined formulation of 5% NaOCl + 40% S. Persica was as effective as conventional protocol and ultrasonic activation improved its efficacy.

Keywords: Ethylenediamine tetraacetic acid, Salvadora persica, sodium hypochlorite, ultrasonic activation


How to cite this article:
Jibin Karim V A, Ranjini M A, Swapna D V, Bharathi M, Nadig RR, Vedavathi B. Evaluation of antimicrobial action, pulp tissue dissolution, and smear layer removal of an experimental irrigant combining Salvadora persica and sodium hypochlorite with and without ultrasonic activation: An in vitro study. Int J Prev Clin Dent Res 2022;9:63-71

How to cite this URL:
Jibin Karim V A, Ranjini M A, Swapna D V, Bharathi M, Nadig RR, Vedavathi B. Evaluation of antimicrobial action, pulp tissue dissolution, and smear layer removal of an experimental irrigant combining Salvadora persica and sodium hypochlorite with and without ultrasonic activation: An in vitro study. Int J Prev Clin Dent Res [serial online] 2022 [cited 2022 Dec 6];9:63-71. Available from: https://www.ijpcdr.org/text.asp?2022/9/3/63/357305




  Introduction Top


Microorganisms and their toxic metabolites are the main causative agents for pulp and peri radicular pathoses. Endodontic therapy aims to eliminate these micro-organisms from the infected root canal system to prevent reinfection.

Irrigation plays a vital role during root canal treatment as it fulfils several important mechanical, chemical, and (micro) biological functions and is also the only way to reach intricate the areas of the root canal system that are left untouched by mechanical instrumentation. An ideal endodontic irrigant must possess pulp tissue dissolution property, broad antimicrobial activity, and smear layer removing ability in addition to the other requirements.[1] Different chemicals have been tried as root canal irrigants with different regimens to achieve these desirable effects.[2]

Sodium hypochlorite (NaOCl) and 17% ethylenediamine tetraacetic acid (EDTA) are being separately used to eliminate both organic and inorganic contents of root canal space. However, the use of multiple irrigants in clinical scenario is time-consuming. Since 17% EDTA has more erosive action on root dentine[3] and lacks any antimicrobial and organic tissue dissolution property, there has been a search for a better chelator material that can be effectively combined with NaOCl as a single irrigant.

Currently herbs are being investigated as irrigants as they have antimicrobial and chelating actions, Salvadora Persica is one among them. Hence, this study was undertaken to evaluate pulp dissolution, antimicrobial property, and smear layer removal using a combined formulation of NaOCl and S. persica, with and without ultrasonic activation.


  Materials and Methods Top


For pulp tissue dissolution

Forty pulp tissue samples weighing 0.9 g were dissolved in each experimental irrigation group

  • Group 1–5 ml of 5% NaOCl
  • Group 2–5 ml of 5% NaOCl with ultrasonic activation
  • Group 3–5 ml of 5% NaOCl + 40% S. persica
  • Group 4–5 ml of 5% NaOCl + 40% S. persica with ultrasonic activation.


Pulp tissue was weighed after the passage time of 1, 5, and 10 min. The difference in weight of the tissue sample before and after exposure to the test solution was divided by the original weight and multiplied by 100 to obtain the percentage of tissue weight loss.

For smear layer removal and energy dispersive X-ray spectroscopy analysis

Forty extracted single-rooted premolars were collected, disinfected, and decoronated at cementoenamel junction to obtain standardized root length of 15 mm. Root canals were instrumented with ProTaper rotary files to F3 size 1 mm short of the radiographic apex. Specimens were divided into four groups according to the irrigation protocol as follows:

  • Group 1 – 5 ml of 5% NaOCl followed by saline and then 1 ml of EDTA for 1 min
  • Group 2 – 5 ml of 5% NaOCl followed by saline and then 1 ml of EDTA with ultrasonic activation.
  • Group 3 – 5 ml of 5% NaOCl +40% S. persica
  • Group 4 – 5 ml of 5% NaOCl +40% S. persica with ultrasonic activation.


Specimens were longitudinally sectioned and evaluated under scanning electron microscope for smear layer removal efficacy and energy dispersive X-ray spectroscopy (EDX) analysis for calcium/phosphate (Ca/PO4) ratio.

For antibacterial activity

Forty extracted single-rooted premolars were collected, decoronated, working length established and instrumented using ProTaper rotary file F3. They were disinfected using autoclave. Root canals were then infected with Enterococcus faecalis and incubated. Irrigation was performed according to various groups as follows:

  • Group 1 – 5 ml of 5% NaOCl followed by saline and then 1 ml of EDTA for 1 min
  • Group 2 – 5 ml of 5% NaOCl followed by saline and then 1 ml of EDTA with ultrasonic activation
  • Group 3 – 5 ml of 5% NaOCl +40% S. persica
  • Group 4 – 5 ml of 5% NaOCl +40% S. persica with ultrasonic activation.


Ultrasonic activation was carried out using Irrisafe tips for two cycles of 30 s each.

After performing the irrigation protocol, dentinal shavings were collected in brain heart infusion (BHI) broth and plated onto BHI agar culture medium and incubated for 48 h. The number of colony-forming units (CFUs) of E. faecalis served as a measure of antibacterial activity.

Statistical analysis

Obtained pulp tissue dissolution values, scanning electron microscopy (SEM) scores and CFU counts were statistically analyzed using Kruskal–Wallis and post hoc Mann–Whitney analysis. One-way ANOVA and post hoc Mann–Whitney analysis for EDX analysis.


  Results Top


Pulp tissue dissolution

Pulp tissue dissolution by freshly mixed 5% NaOCl + 40% S. persica with and without ultrasonic activation performed similar to 5% NaOCl with and without ultrasonic activation [Table 1]. Maximum pulp tissue dissolution occurred within 5 min and ultrasonic activation had an additive effect. However, there was no statistically significant difference between groups [Graph 1].
Table 1: Comparison of the pulp tissue remaining (mg) after 1, 5, and 10 min following different irrigation protocols using Kruskal-Wallis test

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Smear layer removal and calcium/phosphate ratio

All the groups removed the smear layer equally with almost all the dentinal tubules remaining patent [Table 2] and [Figure 1]. No statistically significant difference was seen with respect to smear layer removal among the groups tested indicating that combined experimental formulation containing 5% NaOCl + 40% S. persica with and without ultrasonic activation was as effective as 5% NaOCl and 17% EDTA with and without ultrasonic activation in removing smear layer [Graph 2]. EDX analysis shows that 5% NaOCl in combination with 40% S. persica resulted in higher Ca/PO4 in radicular dentine [Table 3] and [Graph 3], [Graph 4], [Graph 5], [Graph 6], [Graph 7].
Figure 1: Scanning electron microscopy images of coronal, middle, and apical thirds after irrigation with different groups

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Table 2: Comparison of the scanning electron microscopy scores of the smear layer remaining among groups at coronal, middle, and apical third using Kruskal-Wallis

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Table 3: One-way ANOVA for group wise comparison of calcium/phosphate ratio at coronal, middle, and third

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Antimicrobial efficacy against Enterococcus faecalis

Five percentage NaOCl +40% S. persica with and without ultrasonic activation showed better antimicrobial efficacy compared to 5% NaOCl and 17% EDTA with and without ultrasonic activation in terms of mean value. However, no statistically significant difference was seen between the tested groups [Table 4], [Graph 8] and [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6].
Figure 2: Control

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Figure 3: Colony-forming units in Group 1

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Figure 4: Colony-forming units in Group 2

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Figure 5: Colony-forming units in Group 3

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Figure 6: Colony-forming units in Group 4

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Table 4: Mean of colony-forming units in various groups using one-way ANOVA

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  Discussion Top


NaOCl is the most commonly used root canal irrigant. It has very good antimicrobial property and organic tissue dissolving ability, but it does not remove the inorganic component. Hence EDTA, a chelator is often used along with NaOCl, which aids in instrumentation of the root canals by chelating with the calcium component of the tooth.[1]

However, these solutions cannot be combined together, as they form an insoluble precipitate which may block the root canals. Hence, saline is usually advocated intermittent to these two irrigants. Furthermore, when 17% EDTA is used as a final rinse, it removed the smear layer and opens up the dentinal tubules that aid in better root canal sealer penetration-thereby increasing the push out bond strength of the obturating materials.[4] However, studies have shown that 17% EDTA was more erosive and altered the Ca/PO4 ratio of root dentine, thereby affecting the tooth's fracture toughness.[5]

Many techniques have been devised to improve the efficacy of irrigants such as increasing the concentration/contact time, temperature or by combining with other chemicals. Mechanical agitation techniques with the use of master cone gutta percha, endodontic files, and sonic and ultrasonic devices have been popular and are easily dispensable with promising results.[6]

S. persica or Pilu is a herbal extract that has been researched. Studies done by Al-Sabawi et al.,[7] Almas,[8] and Tabatabaei et al.[9] have shown it to have excellent biocompatibility, good antimicrobial property, in addition to organic tissue dissolving and chelating property. It could be combined with NaOCl without any interaction, unlike EDTA. Hence, the purpose of this study is met in this aspect.

Pulp tissue dissolution

The pulp tissue-dissolving capacity of any irrigant depends on the amount of irrigant, type and contact time with the tissues, and mass of the organic tissue.[10],[11] In the present study, these factors were standardized, i.e., the same volume of irrigating solutions, pulp tissue of approximately the same weight (0.9 mg), and contact time of 1, 5, and 10 min with the pulp tissue. The results of the present study showed at 1-, 5- and 10-min immersion of pulp tissue in different irrigating solutions, there was almost 80% reduction in the weight of the pulp tissue from the original weight in all the irrigation groups and protocols. The amount of pulp tissue remaining was least in Group 4, followed by Group 3, Group 2, and Group 1 with a mean score of 0.004, 0.017, 0.019, and 0.024, respectively. However, this difference in their values was not statistically significant (P > 0.05). However, Groups 3 and 4 performed better than Group 1 and 2.

NaOCl is already proven to possess excellent pulp tissue dissolution property.[12] This is evident from previous studies and also in our study Groups 1, 2. With NaOCl being common to all the 4 groups; additional pulp tissue dissolution property seen in Group 3, 4 with S. persica, which has showed an additive action. Studies have proved the presence of heat stable polyphenol tannins in S. persica to have organic tissue dissolving property.[13] Thus, higher pulp tissue dissolution in Groups 3 and 4 can be explained. Another important finding of pulp tissue dissolution being slightly higher in groups with ultrasonic activation (Group 2 and 4) compared to nonactivation groups (Group 1 and 3). Ultrasonic activation creates fluid motion within irrigants, improves the contact, dissolution potential and temperature of the irrigating solution, thereby enhancing the pulp tissue dissolution.[14] There was no enhancement in the pulp tissue dissolution potential seen at 5 and 10 min in all irrigation groups, which could probably be due to unavailability of active chlorine ions as time elapses, which are the ones acting on pulp tissue.

Smear layer removal

Root canal instrumentation produces a layer of organic and inorganic material called the smear layer that may also contain bacteria and their by-products. It can prevent the penetration of intracanal medicaments into dentinal tubules and influence the adaptation of filling materials to canal walls. 7% EDTA, a commonly used chelator that removes smear layer, not only reduces micro hardness in radicular dentin but also increases roughness which leads to altered surface texture when used for longer period and at higher volume.[14],[15],[16],[17] Hence, in the current study, 5 ml of 17% EDTA was used for 1 min.

SEM analysis of the coronal, middle, and apical third of the root specimens showed comparatively less smear layer left in Groups 3 and 4 as against Group 1 and 2 at all the three levels. There was no statistically significant difference between groups as analyzed by post hoc Mann–Whitney test.

EDTA has an excellent chelating property and this is also evident in our group, i.e., Group 1, 2 has shown good smear layer removal property. Groups 3 and 4 have shown an equivalent result. The stearic acid content of S. persica would have chelated with the calcium of the root dentine, breaking the weak hydrogen bonds that link collagen of smear layer to dentine surface.[18] Marending et al. have opined that the removal of organic portion of the dentine by the action of NaOCl, further enhanced the penetrability of chelator that would have resulted in better smear layer removal.[19]

The results also showed that at all the three levels, Groups (2 and 4) with ultrasonic activation further enhanced the smear layer removing ability by causing shear stress in the inorganic particles of the smear layer by acoustic streaming, facilitating its removal. Irrigant delivered by conventional needle in Groups 1 and 3 showed lesser smear layer removal as they could be inserted to a limited depth into the root canals and to the vapor lock effect created by these nonside-vented needles and hindering the access of irrigant.

On the other hand, passively activating the irrigant using ultrasonics would have eliminated this vapor lock effect, thereby improving the access of the irrigating solution to root canal intricacies-aiding in better smear layer removal.[20] Accordingly, Group 4 has shown better smear layer removal than the Group 2. Although there was a slight difference in the mean of scores at all three levels, it was not statistically significant. This implies that the experimental combination works similar to that of conventional irrigation with NaOCl and EDTA.

Energy dispersive X-ray spectroscopy analysis for remaining amount of calcium/phosphate

Any change in the calcium/phosphate ratio of root dentine indicates alteration in the inorganic component that affects the hardness, permeability, and solubility of dentin.[5],[15],[21],[22] Results of this study showed that the amount of remaining Ca/PO4 on the prepared dentinal walls in the coronal third decreased in the following order; Group 3 followed by Group 1with a mean score of 2.305 and 2.288, respectively. Although there was a difference in the mean, it was not statistically significant. In ultrasonic activation groups, Group 4 showed higher Ca/PO4 ratio, compared to Group 3 with a mean score of 1.919 and 1.878 respectively. However, it was not statistically significant. Similar findings were observed in the middle third and apical third.

This indicates that the experimental irrigation combination though removed the smear layer effectively, it did not alter Ca/PO4 ratio.

The study also showed that ultrasonic activation resulted in more mineral loss compared to nonactivation group. This could probably be due to the acoustic streaming which could have enhanced the penetration of irrigant and smear layer dislodgement.[23]

Thus, an irrigation regimen that will ensure adequate disinfection without causing much alteration in root canal dentin structure is needed and this experimental combination can be considered as a safer alternative.

Antimicrobial efficacy against Enterococcus faecalis

E. faecalis is a Gram-positive facultative anaerobic bacterium found in root canal reinfection cases. It was selected for the purpose of the present study because it is most resistant to elimination by disinfecting agents.[24] The results of the present study showed that mean CFU's were least in Group 2 followed Group 4, Group 3, and Group 1 with a mean score of 2, 2.4, 4.8, and 5.2, respectively. Furthermore, the mean CFUs was comparatively lesser in the experimental groups with ultrasonic activation than in groups without activation. In the nonactivation groups, Group 3 was found to perform better than Group 1 with a mean score of 4.8 and 5.2, respectively. However, there was no statistically significant difference between the groups.

Salvadora has antibacterial properties due to its sulphur content and trimethylamine. Sofrata et al. identified a volatile compound: Benzyl isothiocyanate in S. persica extracts exhibited rapid and strong bactericidal effect against Gram-negative bacteria but low effect on Gram-positive bacteria.[13],[25],[26],[27],[28]

Our study indicates that combined formulation of 5% NaOCl +40% S. Persica is as effective as 5% NaOCl and 17% EDTA in terms of antimicrobial, chelating agent and pulp dissolution property and that ultrasonic activation could improve these effects. Thus, the experimental formulation of 5% NaOCl +40% S. persica with ultrasonic activation can be considered as an effective alternative to 17% EDTA and 5% NaOCl. Further long-term in-vitro and in vivo studies are needed to investigate the parameters such as push out bond strength of the obturating materials, dentin microhardness, biocompatibility with the periapical tissues, shelf life, cost, and availability of the material before S. persica can be routinely advocated as an alternative to NaOCl.


  Conclusions Top


Within the limitations of this in vitro study, following conclusions can be drawn:

Pulp tissue dissolution

Maximum pulp tissue dissolution was seen with experimental combination of 5% NaOCl +40% S. persica at 1 and 5 min with ultrasonic activation. However, it was not statistically significant from other groups.

Smear layer removal by scanning electron microscopy analysis and energy dispersive X-ray spectroscopy for Ca/P04 ratio

All the four groups were able to remove the smear layer to an extent that almost all the dentinal tubules remained open, but this was not statistically significant. However, ultrasonic activation groups showed better smear layer removal as against the one's without ultrasonic activation. The experimental combination of 5% NaOCl +40% S. persica retained higher Ca/PO4 in the radicular dentin at different levels.

Antimicrobial efficacy by mean colony-forming units against Enterococcus faecalis

Experimental combination showed less number of E. Faecalis CFU's as against conventional irrigation protocol, with ultrasonic activation giving even better results.

Acknowledgments

Department of Conservative dentistry and endodontics. Dayananda sagar college of dental sciences, Bangalore.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

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



 

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